Modified cells comprising CRISPR systems

ABSTRACT

The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA. The present disclosure provides methods of modulating transcription of a target nucleic acid.

CROSS-REFERENCE

This application is a continuation of PCT/US2020/021213, filed Mar. 5,2020, which claims the benefit of U.S. Provisional Patent ApplicationNo. 62/815,173, filed Mar. 7, 2019, U.S. Provisional Patent ApplicationNo. 62/855,739, filed May 31, 2019, U.S. Provisional Patent ApplicationNo. 62/907,422, filed Sep. 27, 2019, and U.S. Provisional PatentApplication No. 62/948,470, filed Dec. 16, 2019, each of whichapplications is incorporated herein by reference in its entirety.

INTRODUCTION

CRISPR-Cas systems include Cas proteins, which are involved inacquisition, targeting and cleavage of foreign DNA or RNA, and a guideRNA(s), which includes a segment that binds Cas proteins and a segmentthat binds to a target nucleic acid. For example, Class 2 CRISPR-Cassystems comprise a single Cas protein bound to a guide RNA, where theCas protein binds to and cleaves a targeted nucleic acid. Theprogrammable nature of these systems has facilitated their use as aversatile technology for use in modification of target nucleic acid.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“09_BERK-403CON8_SeqList_ST25” created on Aug. 16, 2021 and having asize of 224,019 bytes. The contents of the text file are incorporated byreference herein in their entirety.

SUMMARY

The present disclosure provides RNA-guided CRISPR-Cas effector proteins,nucleic acids encoding same, and compositions comprising same. Thepresent disclosure provides ribonucleoprotein complexes comprising: anRNA-guided CRISPR-Cas effector protein of the present disclosure; and aguide RNA. The present disclosure provides methods of modifying a targetnucleic acid, using an RNA-guided CRISPR-Cas effector protein of thepresent disclosure and a guide RNA. The present disclosure providesmethods of modulating transcription of a target nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the size distribution of complete bacteriophage genomesfrom this study, Lak phage reported recently from a subset of the samesamples and reference sources (all dsDNA genomes from RefSeq v92 andnon-artifactual assemblies >200 kb from (Paez-Espino et al. (2016)Nature 536: 425).

FIG. 1B shows a histogram of the genome size distribution of phage withgenomes >200 kb from this study, Lak, and reference genomes. Box andwhisker plots of tRNA counts per genome as a function of genome size.

FIG. 2 shows a phylogenetic tree constructed using terminase sequencesfrom huge phage genomes of this study and related database sequences.

FIG. 3 shows a model for how phage-encoded capacities could function toredirect the host's translational system to produce phage proteins. Nohuge phage has all of these genes, but many have tRNAs (clover leafshapes) and tRNA synthetases (aaRS). Phage proteins with up to 6ribosomal protein S1 domains occur in a few genomes. The S1 binds mRNAto bring it into the site on the ribosome where it is decoded. Ribosomalprotein S21 (S21) might selectively initiate translation of phage mRNAs,and many sequences have N-terminal extensions that may be involved inbinding RNA (dashed line in ribosome insert, which is based on PDB code6bu8 and pmid: 29247757 for ribosome and S1 structural model). Somephage have initiation factors (IF) and elongation factor G (EF G) andsome have rpL7/L12, which could mediate efficient ribosome binding.Abbreviation: RNA pol, RNA polymerase.

FIG. 4A shows a bacterium-phage interaction involving CRISPR targeting(cell diagram).

FIG. 4B shows the interaction network showing targeting of bacterial(from top to bottom: SEQ ID NOs: 163-164) and phage-encoded (from top tobottom: SEQ ID NOs: 163-164) CRISPR spacers.

FIG. 5 shows ecosystems with phage and some plasmids with >200 kbpgenomes, grouped by sampling site type. Each box represents a phagegenome, and boxes are arranged in order of decreasing genome size; sizerange for each site type is listed to the right. Putative host phylum isindicated based on genome phylogenetic profile, with confirmation byCRISPR targeting (X) or information system gene phylogenetic analyses(T).

FIG. 6A-6R provide amino acid sequences of examples of Cas12Jpolypeptides of the present disclosure.

FIG. 7 provides nucleotide sequences of constant region portions ofCas12J guide RNAs (Depicted as the DNA encoding the RNA). Sequences inbold are the orientation used and/or extrapolated from the workingexamples (see, e.g., the crRNA ‘sequences used’ in Example 3). Sequencesseparated by an “or” are the reverse complement of one another.

FIG. 8 depicts consensus sequences for Cas12J guide RNAs.

FIG. 9 provides the positions of amino acids in RuvC-I, RuvC-II, andRuvC-III domains of Cas12J polypeptides that, when substituted, resultsin a Cas12J polypeptide that binds, but does not cleave, a targetnucleic acid in the presence of a Cas12J guide RNA.

FIG. 10 provides a tree showing various CRISPR-Cas effector proteinfamilies.

FIG. 11A-11C shows the efficiency of transformation plasmid interferenceassay.

FIG. 12A-12B shows a demonstration that Cas12J (e.g., Cas12J-1947455,Cas12J-2071242 and Cas12J-3339380) can cleave linear dsDNA fragmentsguided by a crRNA spacer sequence.

FIG. 13 shows results demonstrating the elucidation of PAM sequences.

FIG. 14A-14C illustrates results from mapping RNA sequences to theCas12J CRISPR loci from pBAS::Cas12J-1947455, pBAS::Cas12J-2071242, andpBAS::Cas12J-3339380.

FIG. 15 depicts Cas12j-2- and Cas12j-3-mediated gene editing in humancells.

FIG. 16A-16B provide maps of the pCas12J-3-hs (FIG. 16A) andpCas12J-2-hs (FIG. 16B) constructs.

FIG. 17A-17G present Table 1, which provides nucleotide sequences of thepCas12J-2-hs and pCas12J-3-hs constructs (from top to bottom: SEQ IDNOs: 161-162).

FIG. 18 depicts trans cleavage of ssDNA by Cas12J activated by bindingto DNA.

FIG. 19A-19F depict data showing that Cas12J (CasΦ) is a bona fideCRISPR-Cas system.

FIG. 20 presents a maximum likelihood phylogenetic tree of type Vsubtypes a-k.

FIG. 21A-21B present crRNA repeat similarity (FIG. 21A) among variousCas12J crRNAs and Cas12J amino acid sequence identity (FIG. 21B) amongvarious Cas12J proteins.

FIG. 22A-22C depict CasΦ-3-mediated protection against plasmidtransformation.

FIG. 23A-23D depict cleavage of DNA by CasΦ.

FIG. 24A-24D depict purification of apo CasΦ (CasΦ protein without guideRNA).

FIG. 25A-25C depict production of staggered cuts by CasΦ.

FIG. 26A-26B depict CasΦ-mediated cleavage of dsDNA and ssDNA.

FIG. 27A-27B depict the results of a cleavage assay comparing targetstrand (TS) and non-target strand (NTS) cleavage efficiency by CasΦ.

FIG. 28A-28B depict data showing that CasΦ cleaves ssDNA, but not RNA,in trans upon activation in cis.

FIG. 29A-29D depict processing of pre-crRNA by CasΦ within the RuvCactive site.

FIG. 30A-30C depict processing of pre-crRNA by CasΦ-1 and by CasΦ-2.

FIG. 31A-31B depict formation of ribonucleoprotein (RNP) complexes with:a) pre-crRNA

FIG. 32A-32C depict CasΦ-mediated enhanced green fluorescent protein(EGFP) disruption in HEK293 cells.

FIG. 33A-33B depict data showing CasΦ-mediate genome editing in humancells.

FIG. 34 presents Table 3, which provides a description of some of theplasmids used in Example 7.

FIG. 35 presents Table 4, which provides guide sequences for experimentsdescribed in Example 7.

FIG. 36 presents Table 5, which provides substrate sequences for invitro experiments described in Example 7.

FIG. 37 presents Table 6, which provides crRNA sequences for in vitroexperiments described in Example 7 (from top to bottom SEQ IDNOs:241-250).

DEFINITIONS

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

By “hybridizable” or “complementary” or “substantially complementary” itis meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence ofnucleotides that enables it to non-covalently bind, i.e. formWatson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,”to another nucleic acid in a sequence-specific, antiparallel, manner(i.e., a nucleic acid specifically binds to a complementary nucleicacid) under the appropriate in vitro and/or in vivo conditions oftemperature and solution ionic strength. Standard Watson-Crickbase-pairing includes: adenine (A) pairing with thymidine (T), adenine(A) pairing with uracil (U), and guanine (G) pairing with cytosine (C)[DNA, RNA]. In addition, for hybridization between two RNA molecules(e.g., dsRNA), and for hybridization of a DNA molecule with an RNAmolecule (e.g., when a DNA target nucleic acid base pairs with a guideRNA, etc.): guanine (G) can also base pair with uracil (U). For example,G/U base-pairing is at least partially responsible for the degeneracy(i.e., redundancy) of the genetic code in the context of tRNA anti-codonbase-pairing with codons in mRNA. Thus, in the context of thisdisclosure, a guanine (G) (e.g., of dsRNA duplex of a guide RNAmolecule; of a guide RNA base pairing with a target nucleic acid, etc.)is considered complementary to both a uracil (U) and to an adenine (A).For example, when a G/U base-pair can be made at a given nucleotideposition of a dsRNA duplex of a guide RNA molecule, the position is notconsidered to be non-complementary, but is instead considered to becomplementary.

Hybridization and washing conditions are well known and exemplified inSambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1therein; and Sambrook, J. and Russell, W., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor (2001). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization.

Hybridization requires that the two nucleic acids contain complementarysequences, although mismatches between bases are possible. Theconditions appropriate for hybridization between two nucleic acidsdepend on the length of the nucleic acids and the degree ofcomplementarity, variables well known in the art. The greater the degreeof complementarity between two nucleotide sequences, the greater thevalue of the melting temperature (Tm) for hybrids of nucleic acidshaving those sequences. For hybridizations between nucleic acids withshort stretches of complementarity (e.g. complementarity over 35 orless, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or lessnucleotides) the position of mismatches can become important (seeSambrook et al., supra, 11.7-11.8). Typically, the length for ahybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotidesor more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotidesor more, 22 nucleotides or more, 25 nucleotides or more, or 30nucleotides or more). Temperature, wash solution salt concentration, andother conditions may be adjusted as necessary according to factors suchas length of the region of complementation and the degree ofcomplementation.

It is understood that the sequence of a polynucleotide need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable or hybridizable. Moreover, a polynucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a bulge, a loop structureor hairpin structure, etc.). A polynucleotide can comprise 60% or more,65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% ormore, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%sequence complementarity to a target region within the target nucleicacid sequence to which it will hybridize. For example, an antisensenucleic acid in which 18 of 20 nucleotides of the antisense compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleotides may be clustered orinterspersed with complementary nucleotides and need not be contiguousto each other or to complementary nucleotides. Percent complementaritybetween particular stretches of nucleic acid sequences within nucleicacids can be determined using any convenient method. Example methodsinclude BLAST programs (basic local alignment search tools) andPowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410;Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), e.g., usingdefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2, 482-489), and the like.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

“Binding” as used herein (e.g. with reference to an RNA-binding domainof a polypeptide, binding to a target nucleic acid, and the like) refersto a non-covalent interaction between macromolecules (e.g., between aprotein and a nucleic acid; between a Cas12J polypeptide/guide RNAcomplex and a target nucleic acid; and the like). While in a state ofnon-covalent interaction, the macromolecules are said to be “associated”or “interacting” or “binding” (e.g., when a molecule X is said tointeract with a molecule Y, it is meant the molecule X binds to moleculeY in a non-covalent manner). Not all components of a binding interactionneed be sequence-specific (e.g., contacts with phosphate residues in aDNA backbone), but some portions of a binding interaction may besequence-specific. Binding interactions are generally characterized by adissociation constant (K_(D)) of less than 10⁻⁶ M, less than 10⁻⁷ M,less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻¹⁰ M, less than 10⁻¹¹M, less than 10⁻¹² M, less than 10⁻¹³ M, less than 10⁻¹⁴ M, or less than10⁻¹⁵ M. “Affinity” refers to the strength of binding, increased bindingaffinity being correlated with a lower K_(D).

By “binding domain” it is meant a protein domain that is able to bindnon-covalently to another molecule. A binding domain can bind to, forexample, a DNA molecule (a DNA-binding domain), an RNA molecule (anRNA-binding domain) and/or a protein molecule (a protein-binding domain)In the case of a protein having a protein-binding domain, it can in somecases bind to itself (to form homodimers, homotrimers, etc.) and/or itcan bind to one or more regions of a different protein or proteins.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains consists of glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains consists ofserine and threonine; a group of amino acids having amide containingside chains consisting of asparagine and glutamine; a group of aminoacids having aromatic side chains consists of phenylalanine, tyrosine,and tryptophan; a group of amino acids having basic side chains consistsof lysine, arginine, and histidine; a group of amino acids having acidicside chains consists of glutamate and aspartate; and a group of aminoacids having sulfur containing side chains consists of cysteine andmethionine. Exemplary conservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine-glycine, and asparagine-glutamine.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same, and inthe same relative position, when comparing the two sequences. Sequenceidentity can be determined in a number of different ways. To determinesequence identity, sequences can be aligned using various convenientmethods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT,etc.), available over the world wide web at sites includingncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/,ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See,e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

A DNA sequence that “encodes” a particular RNA is a DNA nucleotidesequence that is transcribed into RNA. A DNA polynucleotide may encodean RNA (mRNA) that is translated into protein (and therefore the DNA andthe mRNA both encode the protein), or a DNA polynucleotide may encode anRNA that is not translated into protein (e.g. tRNA, rRNA, microRNA(miRNA), a “non-coding” RNA (ncRNA), a guide RNA, etc.).

A “protein coding sequence” or a sequence that encodes a particularprotein or polypeptide, is a nucleotide sequence that is transcribedinto mRNA (in the case of DNA) and is translated (in the case of mRNA)into a polypeptide in vitro or in vivo when placed under the control ofappropriate regulatory sequences.

The terms “DNA regulatory sequences,” “control elements,” and“regulatory elements,” used interchangeably herein, refer totranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, terminators, protein degradationsignals, and the like, that provide for and/or regulate transcription ofa non-coding sequence (e.g., guide RNA) or a coding sequence (e.g.,RNA-guided endonuclease, GeoCas9 polypeptide, GeoCas9 fusionpolypeptide, and the like) and/or regulate translation of an encodedpolypeptide.

As used herein, a “promoter” or a “promoter sequence” is a DNAregulatory region capable of binding RNA polymerase and initiatingtranscription of a downstream (3′ direction) coding or non-codingsequence. For purposes of the present disclosure, the promoter sequenceis bounded at its 3′ terminus by the transcription initiation site andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to initiate transcription at levels detectableabove background. Within the promoter sequence will be found atranscription initiation site, as well as protein binding domainsresponsible for the binding of RNA polymerase. Eukaryotic promoters willoften, but not always, contain “TATA” boxes and “CAT” boxes. Variouspromoters, including inducible promoters, may be used to driveexpression by the various vectors of the present disclosure.

The term “naturally-occurring” or “unmodified” or “wild type” as usedherein as applied to a nucleic acid, a polypeptide, a cell, or anorganism, refers to a nucleic acid, polypeptide, cell, or organism thatis found in nature. For example, a polypeptide or polynucleotidesequence that is present in an organism that can be isolated from asource in nature is naturally occurring.

The term “fusion” as used herein as applied to a nucleic acid orpolypeptide refers to two components that are defined by structuresderived from different sources. For example, where “fusion” is used inthe context of a fusion polypeptide (e.g., a fusion Cas12J protein), thefusion polypeptide includes amino acid sequences that are derived fromdifferent polypeptides. A fusion polypeptide may comprise eithermodified or naturally-occurring polypeptide sequences (e.g., a firstamino acid sequence from a modified or unmodified Cas12J protein; and asecond amino acid sequence from a modified or unmodified protein otherthan a Cas12J protein, etc.). Similarly, “fusion” in the context of apolynucleotide encoding a fusion polypeptide includes nucleotidesequences derived from different coding regions (e.g., a firstnucleotide sequence encoding a modified or unmodified Cas12J protein;and a second nucleotide sequence encoding a polypeptide other than aCas12J protein).

The term “fusion polypeptide” refers to a polypeptide which is made bythe combination (i.e., “fusion”) of two otherwise separated segments ofamino acid sequence, usually through human intervention.

“Heterologous,” as used herein, means a nucleotide or polypeptidesequence that is not found in the native nucleic acid or protein,respectively. For example, in some cases, in a variant Cas12J protein ofthe present disclosure, a portion of naturally-occurring Cas12Jpolypeptide (or a variant thereof) may be fused to a heterologouspolypeptide (i.e. an amino acid sequence from a protein other than aCas12J polypeptide or an amino acid sequence from another organism). Asanother example, a fusion Cas12J polypeptide can comprise all or aportion of a naturally-occurring Cas12J polypeptide (or variant thereof)fused to a heterologous polypeptide, i.e., a polypeptide from a proteinother than a Cas12J polypeptide, or a polypeptide from another organism.The heterologous polypeptide may exhibit an activity (e.g., enzymaticactivity) that will also be exhibited by the variant Cas12J protein orthe fusion Cas12J protein (e.g., biotin ligase activity; nuclearlocalization; etc.). A heterologous nucleic acid sequence may be linkedto a naturally-occurring nucleic acid sequence (or a variant thereof)(e.g., by genetic engineering) to generate a nucleotide sequenceencoding a fusion polypeptide (a fusion protein).

“Recombinant,” as used herein, means that a particular nucleic acid (DNAor RNA) is the product of various combinations of cloning, restriction,polymerase chain reaction (PCR) and/or ligation steps resulting in aconstruct having a structural coding or non-coding sequencedistinguishable from endogenous nucleic acids found in natural systems.DNA sequences encoding polypeptides can be assembled from cDNA fragmentsor from a series of synthetic oligonucleotides, to provide a syntheticnucleic acid which is capable of being expressed from a recombinanttranscriptional unit contained in a cell or in a cell-free transcriptionand translation system. Genomic DNA comprising the relevant sequencescan also be used in the formation of a recombinant gene ortranscriptional unit. Sequences of non-translated DNA may be present 5′or 3′ from the open reading frame, where such sequences do not interferewith manipulation or expression of the coding regions, and may indeedact to modulate production of a desired product by various mechanisms(see “DNA regulatory sequences”). Alternatively, DNA sequences encodingRNA (e.g., guide RNA) that is not translated may also be consideredrecombinant. Thus, e.g., the term “recombinant” nucleic acid refers toone which is not naturally occurring, e.g., is made by the artificialcombination of two otherwise separated segments of sequence throughhuman intervention. This artificial combination is often accomplished byeither chemical synthesis means, or by the artificial manipulation ofisolated segments of nucleic acids, e.g., by genetic engineeringtechniques. Such is usually done to replace a codon with a codonencoding the same amino acid, a conservative amino acid, or anon-conservative amino acid. Alternatively, it is performed to jointogether nucleic acid segments of desired functions to generate adesired combination of functions. This artificial combination is oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques. When a recombinant polynucleotide encodes apolypeptide, the sequence of the encoded polypeptide can be naturallyoccurring (“wild type”) or can be a variant (e.g., a mutant) of thenaturally occurring sequence. An example of such a case is a DNA (arecombinant) encoding a wild-type protein where the DNA sequence iscodon optimized for expression of the protein in a cell (e.g., aeukaryotic cell) in which the protein is not naturally found (e.g.,expression of a CRISPR/Cas RNA-guided polypeptide such as Cas12J (e.g.,wild-type Cas12J; variant Cas12J; fusion Cas12J; etc.) in a eukaryoticcell). A codon-optimized DNA can therefore be recombinant andnon-naturally occurring while the protein encoded by the DNA may have awild type amino acid sequence.

Thus, the term “recombinant” polypeptide does not necessarily refer to apolypeptide whose amino acid sequence does not naturally occur. Instead,a “recombinant” polypeptide is encoded by a recombinant non-naturallyoccurring DNA sequence, but the amino acid sequence of the polypeptidecan be naturally occurring (“wild type”) or non-naturally occurring(e.g., a variant, a mutant, etc.). Thus, a “recombinant” polypeptide isthe result of human intervention, but may have a naturally occurringamino acid sequence.

A “vector” or “expression vector” is a replicon, such as plasmid, phage,virus, artificial chromosome, or cosmid, to which another DNA segment,i.e. an “insert”, may be attached so as to bring about the replicationof the attached segment in a cell.

An “expression cassette” comprises a DNA coding sequence operably linkedto a promoter. “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a coding sequence (or the coding sequence can also be said tobe operably linked to the promoter) if the promoter affects itstranscription or expression.

The terms “recombinant expression vector,” or “DNA construct” are usedinterchangeably herein to refer to a DNA molecule comprising a vectorand an insert. Recombinant expression vectors are usually generated forthe purpose of expressing and/or propagating the insert(s), or for theconstruction of other recombinant nucleotide sequences. The insert(s)may or may not be operably linked to a promoter sequence and may or maynot be operably linked to DNA regulatory sequences.

A cell has been “genetically modified” or “transformed” or “transfected”by exogenous DNA or exogenous RNA, e.g. a recombinant expression vector,when such DNA has been introduced inside the cell. The presence of theexogenous DNA results in permanent or transient genetic change. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clones thatcomprise a population of daughter cells containing the transforming DNA.A “clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

Suitable methods of genetic modification (also referred to as“transformation”) include e.g., viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, calcium phosphate precipitation, polyethyleneimine(PEI)-mediated transfection, DEAE-dextran mediated transfection,liposome-mediated transfection, particle gun technology, calciumphosphate precipitation, direct micro injection, nanoparticle-mediatednucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012Sep. 13. pii: 50169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023),and the like.

The choice of method of genetic modification is generally dependent onthe type of cell being transformed and the circumstances under which thetransformation is taking place (e.g., in vitro, ex vivo, or in vivo). Ageneral discussion of these methods can be found in Ausubel, et al.,Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

A “target nucleic acid” as used herein is a polynucleotide (e.g., DNAsuch as genomic DNA) that includes a site (“target site” or “targetsequence”) targeted by an RNA-guided endonuclease polypeptide (e.g.,wild-type Cas12J; variant Cas12J; fusion Cas12J; etc.). The targetsequence is the sequence to which the guide sequence of a subject Cas12Jguide RNA (e.g., a dual Cas12J guide RNA or a single-molecule Cas12Jguide RNA) will hybridize. For example, the target site (or targetsequence) 5′-GAGCAUAUC-3′ within a target nucleic acid is targeted by(or is bound by, or hybridizes with, or is complementary to) thesequence 5′-GAUAUGCUC-3′. Suitable hybridization conditions includephysiological conditions normally present in a cell. For a doublestranded target nucleic acid, the strand of the target nucleic acid thatis complementary to and hybridizes with the guide RNA is referred to asthe “complementary strand” or “target strand”; while the strand of thetarget nucleic acid that is complementary to the “target strand” (and istherefore not complementary to the guide RNA) is referred to as the“non-target strand” or “non-complementary strand.”

By “cleavage” it is meant the breakage of the covalent backbone of atarget nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiatedby a variety of methods including, but not limited to, enzymatic orchemical hydrolysis of a phosphodiester bond. Both single-strandedcleavage and double-stranded cleavage are possible, and double-strandedcleavage can occur as a result of two distinct single-stranded cleavageevents.

“Nuclease” and “endonuclease” are used interchangeably herein to mean anenzyme which possesses catalytic activity for nucleic acid cleavage(e.g., ribonuclease activity (ribonucleic acid cleavage),deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

By “cleavage domain” or “active domain” or “nuclease domain” of anuclease it is meant the polypeptide sequence or domain within thenuclease which possesses the catalytic activity for nucleic acidcleavage. A cleavage domain can be contained in a single polypeptidechain or cleavage activity can result from the association of two (ormore) polypeptides. A single nuclease domain may consist of more thanone isolated stretch of amino acids within a given polypeptide.

The term “stem cell” is used herein to refer to a cell (e.g., plant stemcell, vertebrate stem cell) that has the ability both to self-renew andto generate a differentiated cell type (see Morrison et al. (1997) Cell88:287-298). In the context of cell ontogeny, the adjective“differentiated”, or “differentiating” is a relative term. A“differentiated cell” is a cell that has progressed further down thedevelopmental pathway than the cell it is being compared with. Thus,pluripotent stem cells (described below) can differentiate intolineage-restricted progenitor cells (e.g., mesodermal stem cells), whichin turn can differentiate into cells that are further restricted (e.g.,neuron progenitors), which can differentiate into end-stage cells (i.e.,terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.),which play a characteristic role in a certain tissue type, and may ormay not retain the capacity to proliferate further. Stem cells may becharacterized by both the presence of specific markers (e.g., proteins,RNAs, etc.) and the absence of specific markers. Stem cells may also beidentified by functional assays both in vitro and in vivo, particularlyassays relating to the ability of stem cells to give rise to multipledifferentiated progeny.

Stem cells of interest include pluripotent stem cells (PSCs). The term“pluripotent stem cell” or “PSC” is used herein to mean a stem cellcapable of producing all cell types of the organism. Therefore, a PSCcan give rise to cells of all germ layers of the organism (e.g., theendoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells arecapable of forming teratomas and of contributing to ectoderm, mesoderm,or endoderm tissues in a living organism. Pluripotent stem cells ofplants are capable of giving rise to all cell types of the plant (e.g.,cells of the root, stem, leaves, etc.).

PSCs of animals can be derived in a number of different ways. Forexample, embryonic stem cells (ESCs) are derived from the inner cellmass of an embryo (Thomson et. al, Science. 1998 Nov. 6;282(5391):1145-7) whereas induced pluripotent stem cells (iPSCs) arederived from somatic cells (Takahashi et. al, Cell. 2007 Nov. 30;131(5):861-72; Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et.al, Science. 2007 Dec. 21; 318(5858):1917-20. Epub 2007 Nov. 20).Because the term PSC refers to pluripotent stem cells regardless oftheir derivation, the term PSC encompasses the terms ESC and iPSC, aswell as the term embryonic germ stem cells (EGSC), which are anotherexample of a PSC. PSCs may be in the form of an established cell line,they may be obtained directly from primary embryonic tissue, or they maybe derived from a somatic cell. PSCs can be target cells of the methodsdescribed herein.

By “embryonic stem cell” (ESC) is meant a PSC that was isolated from anembryo, typically from the inner cell mass of the blastocyst. ESC linesare listed in the NIH Human Embryonic Stem Cell Registry, e.g.hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1,HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1(MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (Universityof California at San Francisco); and H1, H7, H9, H13, H14 (WisconsinAlumni Research Foundation (WiCell Research Institute)). Stem cells ofinterest also include embryonic stem cells from other primates, such asRhesus stem cells and marmoset stem cells. The stem cells may beobtained from any mammalian species, e.g. human, equine, bovine,porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc.(Thomson et al. (1998) Science 282:1145; Thomson et al. (1995) Proc.Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254;Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Inculture, ESCs typically grow as flat colonies with largenucleo-cytoplasmic ratios, defined borders and prominent nucleoli. Inaddition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and AlkalinePhosphatase, but not SSEA-1. Examples of methods of generating andcharacterizing ESCs may be found in, for example, U.S. Pat. Nos.7,029,913, 5,843,780, and 6,200,806, the disclosures of which areincorporated herein by reference. Methods for proliferating hESCs in theundifferentiated form are described in WO 99/20741, WO 01/51616, and WO03/020920.

By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EGcell” is meant a PSC that is derived from germ cells and/or germ cellprogenitors, e.g. primordial germ cells, i.e. those that would becomesperm and eggs. Embryonic germ cells (EG cells) are thought to haveproperties similar to embryonic stem cells as described above. Examplesof methods of generating and characterizing EG cells may be found in,for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al., (1992) Cell70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113;Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; andKoshimizu, U., et al. (1996) Development, 122:1235, the disclosures ofwhich are incorporated herein by reference.

By “induced pluripotent stem cell” or “iPSC” it is meant a PSC that isderived from a cell that is not a PSC (i.e., from a cell this isdifferentiated relative to a PSC). iPSCs can be derived from multipledifferent cell types, including terminally differentiated cells. iPSCshave an ES cell-like morphology, growing as flat colonies with largenucleo-cytoplasmic ratios, defined borders and prominent nuclei. Inaddition, iPSCs express one or more key pluripotency markers known byone of ordinary skill in the art, including but not limited to AlkalinePhosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1,Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods ofgenerating and characterizing iPSCs may be found in, for example, U.S.Patent Publication Nos. US20090047263, US20090068742, US20090191159,US20090227032, US20090246875, and US20090304646, the disclosures ofwhich are incorporated herein by reference. Generally, to generateiPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4,SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram thesomatic cells to become pluripotent stem cells.

By “somatic cell” it is meant any cell in an organism that, in theabsence of experimental manipulation, does not ordinarily give rise toall types of cells in an organism. In other words, somatic cells arecells that have differentiated sufficiently that they will not naturallygenerate cells of all three germ layers of the body, i.e. ectoderm,mesoderm and endoderm. For example, somatic cells would include bothneurons and neural progenitors, the latter of which may be able tonaturally give rise to all or some cell types of the central nervoussystem but cannot give rise to cells of the mesoderm or endodermlineages.

By “mitotic cell” it is meant a cell undergoing mitosis. Mitosis is theprocess by which a eukaryotic cell separates the chromosomes in itsnucleus into two identical sets in two separate nuclei. It is generallyfollowed immediately by cytokinesis, which divides the nuclei,cytoplasm, organelles and cell membrane into two cells containingroughly equal shares of these cellular components.

By “post-mitotic cell” it is meant a cell that has exited from mitosis,i.e., it is “quiescent”, i.e. it is no longer undergoing divisions. Thisquiescent state may be temporary, i.e. reversible, or it may bepermanent.

By “meiotic cell” it is meant a cell that is undergoing meiosis. Meiosisis the process by which a cell divides its nuclear material for thepurpose of producing gametes or spores. Unlike mitosis, in meiosis, thechromosomes undergo a recombination step which shuffles genetic materialbetween chromosomes. Additionally, the outcome of meiosis is four(genetically unique) haploid cells, as compared with the two(genetically identical) diploid cells produced from mitosis.

In some instances, a component (e.g., a nucleic acid component (e.g., aCas12J guide RNA); a protein component (e.g., wild-type Cas12Jpolypeptide; variant Cas12J polypeptide; fusion Cas12J polypeptide;etc.); and the like) includes a label moiety. The terms “label”,“detectable label”, or “label moiety” as used herein refer to any moietythat provides for signal detection and may vary widely depending on theparticular nature of the assay. Label moieties of interest include bothdirectly detectable labels (direct labels; e.g., a fluorescent label)and indirectly detectable labels (indirect labels; e.g., a binding pairmember). A fluorescent label can be any fluorescent label (e.g., afluorescent dye (e.g., fluorescein, Texas red, rhodamine, ALEXAFLUOR®labels, and the like), a fluorescent protein (e.g., green fluorescentprotein (GFP), enhanced GFP (EGFP), yellow fluorescent protein (YFP),red fluorescent protein (RFP), cyan fluorescent protein (CFP), cherry,tomato, tangerine, and any fluorescent derivative thereof), etc.).Suitable detectable (directly or indirectly) label moieties for use inthe methods include any moiety that is detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical,chemical, or other means. For example, suitable indirect labels includebiotin (a binding pair member), which can be bound by streptavidin(which can itself be directly or indirectly labeled). Labels can alsoinclude: a radiolabel (a direct label)(e.g., ³H, ¹²⁵I, ³⁵S, or ³²P); anenzyme (an indirect label)(e.g., peroxidase, alkaline phosphatase,galactosidase, luciferase, glucose oxidase, and the like); a fluorescentprotein (a direct label)(e.g., green fluorescent protein, redfluorescent protein, yellow fluorescent protein, and any convenientderivatives thereof); a metal label (a direct label); a colorimetriclabel; a binding pair member; and the like. By “partner of a bindingpair” or “binding pair member” is meant one of a first and a secondmoiety, wherein the first and the second moiety have a specific bindingaffinity for each other. Suitable binding pairs include, but are notlimited to: antigen/antibodies (for example,digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP,dansyl-X-anti-dansyl, fluorescein/anti-fluorescein, luciferyellow/anti-lucifer yellow, and rhodamine anti-rhodamine), biotin/avidin(or biotin/streptavidin) and calmodulin binding protein(CBP)/calmodulin. Any binding pair member can be suitable for use as anindirectly detectable label moiety.

Any given component, or combination of components can be unlabeled, orcan be detectably labeled with a label moiety. In some cases, when twoor more components are labeled, they can be labeled with label moietiesthat are distinguishable from one another.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, e.g., in a human, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; and (c)relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to an individual organism, e.g., a mammal,including, but not limited to, murines, simians, humans, non-humanprimates, ungulates, felines, canines, bovines, ovines, mammalian farmanimals, mammalian sport animals, and mammalian pets.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aCas12J CRISPR-Cas effector polypeptide” includes a plurality of suchpolypeptides and reference to “the guide RNA” includes reference to oneor more guide RNAs and equivalents thereof known to those skilled in theart, and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides RNA-guided CRISPR-Cas effector proteins,referred to herein as “Cas12J” polypeptides, “CasΦ” polypeptides, or“CasXS” polypeptides”; nucleic acids encoding same; and compositionscomprising same. The present disclosure provides ribonucleoproteincomplexes comprising: a Cas12J polypeptide of the present disclosure;and a guide RNA. The present disclosure provides methods of modifying atarget nucleic acid, using a Cas12J polypeptide of the presentdisclosure and a guide RNA. The present disclosure provides methods ofmodulating transcription of a target nucleic acid.

The present disclosure provides guide RNAs (referred to herein as“Cas12J guide RNAs”) that bind to and provide sequence specificity tothe Cas12J proteins; nucleic acids encoding the Cas12J guide RNAs; andmodified host cells comprising the Cas12J guide RNAs and/or nucleicacids encoding same. Cas12J guide RNAs are useful in a variety ofapplications, which are provided.

Compositions

Crispr/Cas12J Proteins and Guide RNAs

A Cas12J CRISPR/Cas effector polypeptide (e.g., a Cas12J protein; alsoreferred to as a “CasXS polypeptide” or a “CasΦ polypeptide”) interactswith (binds to) a corresponding guide RNA (e.g., a Cas12J guide RNA) toform a ribonucleoprotein (RNP) complex that is targeted to a particularsite in a target nucleic acid (e.g. a target DNA) via base pairingbetween the guide RNA and a target sequence within the target nucleicacid molecule. A guide RNA includes a nucleotide sequence (a guidesequence) that is complementary to a sequence (the target site) of atarget nucleic acid. Thus, a Cas12J protein forms a complex with aCas12J guide RNA and the guide RNA provides sequence specificity to theRNP complex via the guide sequence. The Cas12J protein of the complexprovides the site-specific activity. In other words, the Cas12J proteinis guided to a target site (e.g., stabilized at a target site) within atarget nucleic acid sequence (e.g. a chromosomal sequence or anextrachromosomal sequence, e.g., an episomal sequence, a minicirclesequence, a mitochondrial sequence, a chloroplast sequence, etc.) byvirtue of its association with the guide RNA.

In some cases, a Cas12J CRISPR/Cas effector polypeptide of the presentdisclosure, when complexed with a guide RNA, cleaves double-stranded DNAor single-stranded DNA, but not single-stranded RNA.

In some cases, a Cas12J CRISPR/Cas effector polypeptide of the presentdisclosure catalyzes processing of pre-crRNA in a magnesium-dependentmanner.

The present disclosure provides compositions comprising a Cas12Jpolypeptide (and/or a nucleic acid comprising a nucleotide sequenceencoding the Cas12J polypeptide) (e.g., where the Cas12J polypeptide canbe a naturally existing protein, a nickase Cas12J protein, acatalytically inactive (“dead” Cas12J; also referred to herein as a“dCas12J protein”), a fusion Cas12J protein, etc.). The presentdisclosure provides compositions comprising a Cas12J guide RNA (and/or anucleic acid comprising a nucleotide sequence encoding the Cas12J guideRNA). The present disclosure provides compositions comprising (a) aCas12J polypeptide (and/or a nucleic acid encoding the Cas12Jpolypeptide) (e.g., where the Cas12J polypeptide can be a naturallyexisting protein, a nickase Cas12J protein, a dCas12J protein, a fusionCas12J protein, etc.) and (b) a Cas12J guide RNA (and/or a nucleic acidencoding the Cas12J guide RNA). The present disclosure provides anucleic acid/protein complex (RNP complex) comprising: (a) a Cas12Jpolypeptide of the present disclosure (e.g., where the Cas12Jpolypeptide can be a naturally existing protein, a nickase Cas12Jprotein, a Cdas12J protein, a fusion Cas12J protein, etc.); and (b) aCas12J guide RNA.

Cas12J Protein

A Cas12J polypeptide (this term is used interchangeably with the term“Cas12J protein”, “CasΦ polypeptide”, and “CasΦ protein”) can bindand/or modify (e.g., cleave, nick, methylate, demethylate, etc.) atarget nucleic acid and/or a polypeptide associated with target nucleicacid (e.g., methylation or acetylation of a histone tail) (e.g., in somecases, the Cas12J protein includes a fusion partner with an activity,and in some cases, the Cas12J protein provides nuclease activity). Insome cases, the Cas12J protein is a naturally-occurring protein (e.g.,naturally occurs in bacteriophage). In other cases, the Cas12J proteinis not a naturally-occurring polypeptide (e.g., the Cas12J protein is avariant Cas12J protein (e.g., a catalytically inactive Cas12J protein, afusion Cas12J protein, and the like).

A Cas12J polypeptide (e.g., not fused to any heterologous fusionpartner) can have a molecular weight of from about 65 kiloDaltons (kDa)to about 85 kDa. For example, a Cas12J polypeptide can have a molecularweight of from about 65 kDa to about 70 kDa, from about 70 kDa to about75 kDa, or from about 75 kDa to about 80 kDa. For example, a Cas12Jpolypeptide can have a molecular weight of from about 70 kDa to about 80kDa.

Assays to determine whether given protein interacts with a Cas12J guideRNA can be any convenient binding assay that tests for binding between aprotein and a nucleic acid. Suitable binding assays (e.g., gel shiftassays) will be known to one of ordinary skill in the art (e.g., assaysthat include adding a Cas12J guide RNA and a protein to a target nucleicacid). Assays to determine whether a protein has an activity (e.g., todetermine if the protein has nuclease activity that cleaves a targetnucleic acid and/or some heterologous activity) can be any convenientassay (e.g., any convenient nucleic acid cleavage assay that tests fornucleic acid cleavage). Suitable assays (e.g., cleavage assays) will beknown to one of ordinary skill in the art.

A naturally occurring Cas12J protein functions as an endonuclease thatcatalyzes a double strand break at a specific sequence in a targeteddouble stranded DNA (dsDNA). The sequence specificity is provided by theassociated guide RNA, which hybridizes to a target sequence within thetarget DNA. The naturally occurring Cas12J guide RNA is a crRNA, wherethe crRNA includes (i) a guide sequence that hybridizes to a targetsequence in the target DNA and (ii) a protein binding segment whichincludes a stem-loop (hairpin-dsRNA duplex) that binds to the Cas12Jprotein.

In some cases, a C12J polypeptide of the present disclosure, whencomplexed with a Cas12J guide RNA, generates a product nucleic acidcomprising 5′ overhang following site specific cleavage of a targetnucleic acid. The 5′ overhang can be an 8 to 12 nucleotide (nt)overhang. For example, the 5′ overhang can be 8 nt, 9 nt, 10 nt, 11, nt,or 12 nt in length.

In some embodiments, the Cas12J protein of the subject methods and/orcompositions is (or is derived from) a naturally occurring (wild type)protein. Examples of naturally occurring Cas12J proteins are depicted inFIG. 6A-6R. In some cases, a Cas12J protein (of the subject compositionsand/or methods) includes an amino acid sequence having 20% or moresequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% ormore, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withany one of the Cas12J amino acid sequences depicted in FIG. 6 (e.g., anyone of FIG. 6A-6R). In some cases, a Cas12J protein (of the subjectcompositions and/or methods) includes an amino acid sequence depicted inFIG. 6 (e.g., any one of FIG. 6A-6R).

In some cases, a Cas12J protein (of the subject compositions and/ormethods) has more sequence identity to an amino acid sequence depictedin FIG. 6 (e.g., any of the Cas12J amino acid sequences depicted in FIG.6) than to any of the following: Cas12a proteins, Cas12b proteins,Cas12c proteins, Cas12d proteins, Cas12e proteins, Cas12 g proteins,Cas12h proteins, and Cas12i proteins. In some cases, a Cas12J protein(of the subject compositions and/or methods) includes an amino acidsequence having a RuvC domain (which includes the RuvC-I, RuvC-II, andRuvC-III domains) that has more sequence identity to the RuvC domain ofan amino acid sequence depicted in FIG. 6 (e.g., the RuvC domain of anyof the Cas12J amino acid sequences depicted in FIG. 6) than to the RuvCdomain of any of the following: Cas12a proteins, Cas12b proteins, Cas12cproteins, Cas12d proteins, Cas12e proteins, Cas12 g proteins, Cas12hproteins, and Cas12i proteins.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with the RuvCdomain (which includes the RuvC-I, RuvC-II, and RuvC-III domains) of anyone of the Cas12J amino acid sequences depicted in FIG. 6 (e.g., any oneof FIG. 6A-6R). In some cases, a Cas12J protein (of the subjectcompositions and/or methods) includes an amino acid sequence having 70%or more sequence identity (e.g., 75% or more, 80% or more, 85% or more,90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with the RuvC domain (which includes the RuvC-I,RuvC-II, and RuvC-III domains) of any one of the Cas12J amino acidsequences depicted in FIG. 6 (e.g., any one of FIG. 6A-6R). In somecases, a Cas12J protein (of the subject compositions and/or methods)includes the RuvC domain (which includes the RuvC-I, RuvC-II, andRuvC-III domains) of any one of the Cas12J amino acid sequences depictedin FIG. 6 (e.g., any one of FIG. 6A-6R).

In some cases, a guide RNA that binds a Cas12J polypeptide includes anucleotide sequence depicted in FIG. 7 (or in some cases the reversecomplement of same). In some cases, the guide RNA comprises thenucleotide sequence (N)nX or the reverse complement of same, where N isany nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20, from17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, orfrom 25 to 30), and X is any one of the nucleotide sequences depicted inFIG. 7 (or in some cases the reverse complement of same).

In some cases, a guide RNA that binds a Cas12J polypeptide includes anucleotide sequence having 20% or more sequence identity (e.g., 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with any one of the sequences depictedin FIG. 7 (or in some cases the reverse complement of same). In somecases, the guide RNA comprises the nucleotide sequence (N)nX or thereverse complement of same, where N is any nucleotide, n is an integerfrom 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17 to 22, from18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30), and X anucleotide sequence having 20% or more sequence identity (e.g., 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with any one of the sequences depictedin FIG. 7.

In some cases, a guide RNA that binds a Cas12J polypeptide includes anucleotide sequence having 85% or more sequence identity (e.g., 90% ormore, 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with any one of the sequences depicted in FIG. 7 (orin some cases the reverse complement of same). In some cases, the guideRNA comprises the nucleotide sequence (N)nX or the reverse complement ofsame, where N is any nucleotide, n is an integer from 15 to 30 (e.g.,from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to20, from 20 to 25, or from 25 to 30), and X a nucleotide sequence having85% or more sequence identity (e.g., 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with any oneof the sequences depicted in FIG. 7.

In some cases, a guide RNA that binds a Cas12J polypeptide includes anucleotide sequence depicted in FIG. 7 (or in some cases the reversecomplement of same). In some cases, the guide RNA comprises thenucleotide sequence X(N)n, where N is any nucleotide, n is an integerfrom 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17 to 22, from18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30), and X is anyone of the nucleotide sequences depicted in FIG. 7 (or in some cases thereverse complement of same).

In some cases, a guide RNA that binds a Cas12J polypeptide includes anucleotide sequence having 20% or more sequence identity (e.g., 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with any one of the sequences depictedin FIG. 7 (or in some cases the reverse complement of same). In somecases, the guide RNA comprises the nucleotide sequence X(N)n, where N isany nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20, from17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, orfrom 25 to 30), and X a nucleotide sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with any oneof the sequences depicted in FIG. 7.

Examples of Cas12J proteins are depicted in FIG. 6A-6R. As noted above,a Cas12J polypeptide is also referred to herein as a “CasΦ polypeptide.”For example:

1) the Cas12J polypeptide designated “Cas12J_1947455” (or“Cas12J_1947455_11” in FIG. 9) and depicted in FIG. 6A is also referredto herein as “CasΦ-1”;

2) the Cas12J polypeptide designated “Cas12J_2071242” and depicted inFIG. 6B is also referred to herein as “CasΦ-2”

3) the Cas12J polypeptide designated “Cas12J_3339380 (or“Cas12J_3339380_12” in FIG. 9) and depicted in FIG. 6D is also referredto herein as “CasΦ-3”;

4) the Cas12J polypeptide designated “Cas12J_3877103_16” and depicted inFIG. 6Q is also referred to herein as “CasΦ-4”;

5) the Cas12J polypeptide designated “Cas12J_10000002_47” or“Cas12J_1000002_112” and depicted in FIG. 6G is also referred to hereinas “CasΦ-5”;

6) the Cas12J polypeptide designated “Cas12J_10100763_4” and depicted inFIG. 6H is also referred to herein as “CasΦ-6”;

7) the Cas12J polypeptide designated “Cas12J_1000007_143” or“Cas12J_1000001_267” and depicted in FIG. 6P is also referred to hereinas “CasΦ-7”;

8) the Cas12J polypeptide designated “Cas12J_10000286_53” and depictedin FIG. 6L (or “Cas12J_10000506_8” and depicted in FIG. 6O) is alsoreferred to herein as “CasΦ-8”;

9) the Cas12J polypeptide designated “Cas12J_10001283_7” and depicted inFIG. 6M is also referred to herein as “CasΦ-9”;

10) the Cas12J polypeptide designated “Cas12J_10037042_3” and depictedin FIG. 6E is also referred to herein as “CasΦ-10”.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6A and designated“Cas12J_1947455.” For example, in some cases, a Cas12J protein includesan amino acid sequence having 50% or more sequence identity (e.g., 60%or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100% sequence identity)with the Cas12J amino acid sequence depicted in FIG. 6A. In some cases,a Cas12J protein includes an amino acid sequence having 80% or moresequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6A. In some cases, a Cas12Jprotein includes an amino acid sequence having 90% or more sequenceidentity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or100% sequence identity) with the Cas12J amino acid sequence depicted inFIG. 6A. In some cases, a Cas12J protein includes an amino acid sequencehaving the Cas12J protein sequence depicted in FIG. 6A. In some cases, aCas12J protein includes an amino acid sequence having the Cas12J proteinsequence depicted in FIG. 6A, with the exception that the sequenceincludes an amino acid substitution (e.g., 1, 2, or 3 amino acidsubstitutions) that reduces the naturally occurring catalytic activityof the protein. In some cases, the Cas12J polypeptide has a length offrom 680 amino acids (aa) to 720 aa, e.g., from 680 aa to 690 aa, from690 aa to 700 aa, from 700 aa to 710 aa, or from 710 aa to 720 aa). Insome cases, the Cas12J polypeptide has a length of 707 amino acids. Insome cases, a guide RNA that binds a Cas12J polypeptide (e.g., a Cas12Jpolypeptide comprising an amino acid sequence having 20% or more, 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% ormore, or 100%, amino acid sequence identity to the Cas12J amino acidsequence depicted in FIG. 6A.) includes the following nucleotidesequence: GTCTCGACTAATCGAGCAATCGTTTGAGATCTCTCC (SEQ ID NO: 1) or thereverse complement of same. In some cases, the guide RNA comprises thenucleotide sequence (N)nGTCTCGACTAATCGAGCAATCGTTTGAGATCTCTCC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n isan integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30). TheCas12J protein designated Cas12J_1947455 (or Cas12J_1947455_11 in FIG.9), and depicted in FIG. 6A, is also referred to herein as “ortholog #1”or “Cas12Φ-1.”

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6B and designated“Cas12J_071242.” For example, in some cases, a Cas12J protein includesan amino acid sequence having 50% or more sequence identity (e.g., 60%or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100% sequence identity)with the Cas12J amino acid sequence depicted in FIG. 6B. In some cases,a Cas12J protein includes an amino acid sequence having 80% or moresequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6B. In some cases, a Cas12Jprotein includes an amino acid sequence having 90% or more sequenceidentity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or100% sequence identity) with the Cas12J amino acid sequence depicted inFIG. 6B. In some cases, a Cas12J protein includes an amino acid sequencehaving the Cas12J protein sequence depicted in FIG. 6B. In some cases, aCas12J protein includes an amino acid sequence having the Cas12J proteinsequence depicted in FIG. 6B, with the exception that the sequenceincludes an amino acid substitution (e.g., 1, 2, or 3 amino acidsubstitutions) that reduces the naturally occurring catalytic activityof the protein. In some cases, the Cas12J polypeptide has a length offrom 740 amino acids (aa) to 780 aa, e.g., from 740 aa to 750 aa, from750 aa to 760 aa, from 760 aa to 770 aa, or from 770 aa to 780 aa). Insome cases, the Cas12J polypeptide has a length of 757 amino acids. Insome cases, a guide RNA that binds a Cas12J polypeptide (e.g., a Cas12Jpolypeptide comprising an amino acid sequence having 20% or more, 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% ormore, or 100%, amino acid sequence identity to the Cas12J amino acidsequence depicted in FIG. 6B) includes the following nucleotidesequence: GTCGGAACGCTCAACGATTGCCCCTCACGAGGGGAC (SEQ ID NO: 3) or thereverse complement of same. In some cases, the guide RNA comprises thenucleotide sequence (N)nGTCGGAACGCTCAACGATTGCCCCTCACGAGGGGAC (SEQ ID NO:4) or the reverse complement of same, where N is any nucleotide and n isan integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30). TheCas12J protein designated Cas12J_2071242, and depicted in FIG. 6B, isalso referred to herein as “ortholog #2” or “Cas12Φ-2.”

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6C and designated“Cas12J_1973640.” For example, in some cases, a Cas12J protein includesan amino acid sequence having 50% or more sequence identity (e.g., 60%or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100% sequence identity)with the Cas12J amino acid sequence depicted in FIG. 6C. In some cases,a Cas12J protein includes an amino acid sequence having 80% or moresequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6C. In some cases, a Cas12Jprotein includes an amino acid sequence having 90% or more sequenceidentity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or100% sequence identity) with the Cas12J amino acid sequence depicted inFIG. 6C. In some cases, a Cas12J protein includes an amino acid sequencehaving the Cas12J protein sequence depicted in FIG. 6C. In some cases, aCas12J protein includes an amino acid sequence having the Cas12J proteinsequence depicted in FIG. 6C, with the exception that the sequenceincludes an amino acid substitution (e.g., 1, 2, or 3 amino acidsubstitutions) that reduces the naturally occurring catalytic activityof the protein. In some cases, the Cas12J polypeptide has a length offrom 740 amino acids (aa) to 780 aa, e.g., from 740 aa to 750 aa, from750 aa to 760 aa, from 760 aa to 770 aa, or from 770 aa to 780 aa). Insome cases, the Cas12J polypeptide has a length of 765 amino acids.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6D and designated“Cas12J_3339380.” For example, in some cases, a Cas12J protein includesan amino acid sequence having 50% or more sequence identity (e.g., 60%or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100% sequence identity)with the Cas12J amino acid sequence depicted in FIG. 6D. In some cases,a Cas12J protein includes an amino acid sequence having 80% or moresequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6D. In some cases, a Cas12Jprotein includes an amino acid sequence having 90% or more sequenceidentity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or100% sequence identity) with the Cas12J amino acid sequence depicted inFIG. 6D. In some cases, a Cas12J protein includes an amino acid sequencehaving the Cas12J protein sequence depicted in FIG. 6D. In some cases, aCas12J protein includes an amino acid sequence having the Cas12J proteinsequence depicted in FIG. 6D, with the exception that the sequenceincludes an amino acid substitution (e.g., 1, 2, or 3 amino acidsubstitutions) that reduces the naturally occurring catalytic activityof the protein. In some cases, the Cas12J polypeptide has a length offrom 740 amino acids (aa) to 780 aa, e.g., from 740 aa to 750 aa, from750 aa to 760 aa, from 760 aa to 770 aa, or from 770 aa to 780 aa). Insome cases, the Cas12J polypeptide has a length of 766 amino acids. Insome cases, a guide RNA that binds a Cas12J polypeptide (e.g., a Cas12Jpolypeptide comprising an amino acid sequence having 20% or more, 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% ormore, or 100%, amino acid sequence identity to the Cas12J amino acidsequence depicted in FIG. 6D) includes the following nucleotidesequence: GTCCCAGCGTACTGGGCAATCAATAGTCGTTTTGGT (SEQ ID NO: 5) or thereverse complement of same. In some cases, the guide RNA comprises thenucleotide sequence (N)nGTCCCAGCGTACTGGGCAATCAATAGTCGTTTTGGT (SEQ ID NO:6) or the reverse complement of same, where N is any nucleotide and n isan integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30). TheCas12J protein designated Cas12J_3339380, and depicted in FIG. 6D, isalso referred to herein as “ortholog #3” or “Cas12Φ-3.”

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6E and designated“Cas12J_10037042_3.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6E. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6E. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6E. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6E. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6E, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 780 amino acids (aa) to 820 aa, e.g., from 780 aa to 790aa, from 790 aa to 800 aa, from 800 aa to 810 aa, or from 810 aa to 820aa). In some cases, the Cas12J polypeptide has a length of 812 aminoacids.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6F and designated“Cas12J_10020921_9.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6F. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6F. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6F. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6F. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6F, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 780 amino acids (aa) to 820 aa, e.g., from 780 aa to 790aa, from 790 aa to 800 aa, from 800 aa to 810 aa, or from 810 aa to 820aa). In some cases, the Cas12J polypeptide has a length of 812 aminoacids.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6G and designated“Cas12J_10000002_47.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6G. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6G. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6G. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6G. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6G, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 770 amino acids (aa) to 810 aa, e.g., from 770 aa to 780aa, from 780 aa to 790 aa, from 790 aa to 800 aa, or from 800 aa to 810aa). In some cases, the Cas12J polypeptide has a length of 793 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6G) includes the followingnucleotide sequence: GGATCCAATCCTTTTTGATTGCCCAATTCGTTGGGAC (SEQ ID NO:7) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence(N)nGGATCCAATCCTTTTTGATTGCCCAATTCGTTGGGAC (SEQ ID NO: 8) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6H and designated“Cas12J_10100763_4.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6H. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6H. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6H. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6H. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6H, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 420 amino acids (aa) to 460 aa, e.g., from 420 aa to 430aa, from 430 aa to 440 aa, from 440 aa to 450 aa, or from 450 aa to 460aa). In some cases, the Cas12J polypeptide has a length of 441 aminoacids.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6I and designated“Cas12J_10004149_10.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6I. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6I. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6I. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6I. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6I, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 790 amino acids (aa) to 830 aa, e.g., from 790 aa to 800aa, from 800 aa to 810 aa, from 810 aa to 820 aa, or rom 820 aa to 830aa). In some cases, the Cas12J polypeptide has a length of 812 aminoacids.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6J and designated“Cas12J_10000724_71.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6J. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6J. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6J. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6J. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6J, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 790 amino acids (aa) to 830 aa, e.g., from 790 aa to 800aa, from 800 aa to 810 aa, from 810 aa to 820 aa, or from 820 aa to 830aa). In some cases, the Cas12J polypeptide has a length of 812 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6J) includes the followingnucleotide sequence: GGATCTGAGGATCATTATTGCTCGTTACGACGAGAC (SEQ ID NO: 9)or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence(N)nGGATCTGAGGATCATTATTGCTCGTTACGACGAGAC (SEQ ID NO: 10) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30. In some cases, aguide RNA that binds a Cas12J polypeptide (e.g., a Cas12J polypeptidecomprising an amino acid sequence having 20% or more, 30% or more, 40%or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100%, amino acid sequence identity to the Cas12J amino acid sequencedepicted in FIG. 6J) includes the following nucleotide sequence:GTCTCGTCGTAACGAGCAATAATGATCCTCAGATCC (SEQ ID NO: 11) or the reversecomplement of same. In some cases, the guide RNA comprises thenucleotide sequence (N)n GTCTCGTCGTAACGAGCAATAATGATCCTCAGATCC (SEQ IDNO: 12) or the reverse complement of same, where N is any nucleotide andn is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6K and designated“Cas12J_1000001_267.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6K. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6K. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6K. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6K. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6K, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 750 amino acids (aa) to 790 aa, e.g., from 750 aa to 760aa, from 760 aa to 770 aa, from 770 aa to 780 aa, or from 780 aa to 790aa). In some cases, the Cas12J polypeptide has a length of 772 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6K) includes the followingnucleotide sequence: GTCTCAGCGTACTGAGCAATCAAAAGGTTTCGCAGG (SEQ ID NO:13) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence(N)nGTCTCAGCGTACTGAGCAATCAAAAGGTTTCGCAGG (SEQ ID NO: 14) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6L and designated“Cas12J_10000286_53.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6L. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6L. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6L. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6L. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6L, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 700 amino acids (aa) to 740 aa, e.g., from 700 aa to 710aa, from 710 aa to 720 aa, from 720 aa to 730 aa, or from 730 aa to 740aa). In some cases, the Cas12J polypeptide has a length of 717 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6L) includes the followingnucleotide sequence: GTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ ID NO:15) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence(N)nGTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ ID NO: 16) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6M and designated“Cas12J_10001283_7.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6M. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6M. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6M. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6M. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6M, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 770 amino acids (aa) to 810 aa, e.g., from 770 aa to 780aa, from 780 aa to 790 aa, from 790 aa to 800 aa, or from 800 aa to 810aa). In some cases, the Cas12J polypeptide has a length of 793 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6M) includes the followingnucleotide sequence: GTCTCGGCGCACCGAGCAATCAGCGAGGTCTTCTAC (SEQ ID NO:17) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence(N)nGTCTCGGCGCACCGAGCAATCAGCGAGGTCTTCTAC (SEQ ID NO: 18) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6N and designated“Cas12J_1000002_112.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6N. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6N. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6N. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6N. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6N, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 770 amino acids (aa) to 810 aa, e.g., from 770 aa to 780aa, from 780 aa to 790 aa, from 790 aa to 800 aa, or from 800 aa to 810aa). In some cases, the Cas12J polypeptide has a length of 793 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6N) includes the followingnucleotide sequence: GTCCCAACGAATTGGGCAATCAAAAAGGATTGGATCC (SEQ ID NO:19) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence(N)nGTCCCAACGAATTGGGCAATCAAAAAGGATTGGATCC (SEQ ID NO: 20) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6O and designated“Cas12J_10000506_8.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6O. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6O. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6O. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6O. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6O, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 700 amino acids (aa) to 740 aa, e.g., from 700 aa to 710aa, from 710 aa to 720 aa, from 720 aa to 730 aa, or from 730 aa to 740aa). In some cases, the Cas12J polypeptide has a length of 717 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6O) includes the followingnucleotide sequence: GTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ ID NO:15) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence(N)nGTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ ID NO: 16) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6P and designated“Cas12J_1000007_143.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6P. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6P. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6P. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6P. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6P, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 750 amino acids (aa) to 790 aa, e.g., from 750 aa to 760aa, from 760 aa to 770 aa, from 770 aa to 780 aa, or from 780 aa to 790aa). In some cases, the Cas12J polypeptide has a length of 772 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6P) includes the followingnucleotide sequence: GTCTCAGCGTACTGAGCAATCAAAAGGTTTCGCAGG (SEQ ID NO:13) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence(N)nGTCTCAGCGTACTGAGCAATCAAAAGGTTTCGCAGG (SEQ ID NO: 14) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6Q and designated“Cas12J_3877103_16.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6Q. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6Q. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6Q. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6Q. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6Q, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 750 amino acids (aa) to 790 aa, e.g., from 750 aa to 760aa, from 760 aa to 770 aa, from 770 aa to 780 aa, or from 780 aa to 790aa). In some cases, the Cas12J polypeptide has a length of 765 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6Q) includes the followingnucleotide sequence: GTCGCGGCGTACCGCGCAATGAGAGTCTGTTGCCAT (SEQ ID NO:21) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence (N)nGTCGCGGCGTACCGCGCAATGAGAGTCTGTTGCCAT (SEQ ID NO: 22) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

In some cases, a Cas12J protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with theCas12J amino acid sequence depicted in FIG. 6R and designated“Cas12J_877636_12.” For example, in some cases, a Cas12J proteinincludes an amino acid sequence having 50% or more sequence identity(e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the Cas12J amino acid sequence depicted in FIG. 6R. Insome cases, a Cas12J protein includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,97% or more, 98% or more, 99% or more, or 100% sequence identity) withthe Cas12J amino acid sequence depicted in FIG. 6R. In some cases, aCas12J protein includes an amino acid sequence having 90% or moresequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100% sequence identity) with the Cas12J amino acid sequencedepicted in FIG. 6R. In some cases, a Cas12J protein includes an aminoacid sequence having the Cas12J protein sequence depicted in FIG. 6R. Insome cases, a Cas12J protein includes an amino acid sequence having theCas12J protein sequence depicted in FIG. 6R, with the exception that thesequence includes an amino acid substitution (e.g., 1, 2, or 3 aminoacid substitutions) that reduces the naturally occurring catalyticactivity of the protein. In some cases, the Cas12J polypeptide has alength of from 750 amino acids (aa) to 790 aa, e.g., from 750 aa to 760aa, from 760 aa to 770 aa, from 770 aa to 780 aa, or from 780 aa to 790aa). In some cases, the Cas12J polypeptide has a length of 766 aminoacids. In some cases, a guide RNA that binds a Cas12J polypeptide (e.g.,a Cas12J polypeptide comprising an amino acid sequence having 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, or 100%, amino acid sequence identity to the Cas12Jamino acid sequence depicted in FIG. 6R) includes the followingnucleotide sequence: ACCAAAACGACTATTGATTGCCCAGTACGCTGGGAC (SEQ ID NO:23) or the reverse complement of same. In some cases, the guide RNAcomprises the nucleotide sequence (N)nACCAAAACGACTATTGATTGCCCAGTACGCTGGGAC (SEQ ID NO: 24) or the reversecomplement of same, where N is any nucleotide and n is an integer from15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to22, from 18 to 20, from 20 to 25, or from 25 to 30.

Cas12J Variants

A variant Cas12J protein has an amino acid sequence that is different byat least one amino acid (e.g., has a deletion, insertion, substitution,fusion) when compared to the amino acid sequence of the correspondingwild type Cas12J protein, e.g., when compared to the Cas12J amino acidsequence depicted in any one of FIG. 6A-6R. In some cases, a Cas12Jvariant comprises from 1 amino acid substitution to 10 amino acidsubstitutions compared to the Cas12J amino acid sequence depicted in anyone of FIG. 6A-6R. In some cases, a Cas12J variant comprises from 1amino acid substitution to 10 amino acid substitutions in the RuvCdomain, compared to the Cas12J amino acid sequence depicted in any oneof FIG. 6A-6R.

Variants—Catalytic Activity

In some cases, the Cas12J protein is a variant Cas12J protein, e.g.,mutated relative to the naturally occurring catalytically activesequence, and exhibits reduced cleavage activity (e.g., exhibits 90%, orless, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less,or 30% or less cleavage activity) when compared to the correspondingnaturally occurring sequence. In some cases, such a variant Cas12Jprotein is a catalytically ‘dead’ protein (has substantially no cleavageactivity) and can be referred to as a ‘dCas12J.’ In some cases, thevariant Cas12J protein is a nickase (cleaves only one strand of a doublestranded target nucleic acid, e.g., a double stranded target DNA). Asdescribed in more detail herein, in some cases, a Cas12J protein (insome case a Cas12J protein with wild type cleavage activity and in somecases a variant Cas12J with reduced cleavage activity, e.g., a dCas12Jor a nickase Cas12J) is fused (conjugated) to a heterologous polypeptidethat has an activity of interest (e.g., a catalytic activity ofinterest) to form a fusion protein (a fusion Cas12J protein).

Amino acid substitutions that result in a Cas12J polypeptide that, whencomplexed with a Cas12J guide RNA, binds, but does not cleave, a targetnucleic acid are depicted in FIG. 9. For example, a substitution of theAsp at position 464 of Cas12J_10037042_3, or a corresponding position inanother Cas12J, results in a dCas12J. As another example, a substitutionof the Glu at position 678 of Cas12J_10037042_3, or a correspondingposition in another Cas12J, results in a dCas12J. As another example, asubstation of the Asp at position 769 of Cas12J_10037042_3, or acorresponding position in another Cas12J, results in a dCas12J.

An amino acid substitution that results in a dCas12J polypeptide (i.e.,a Cas12J polypeptide that binds, but does not cleave, a target nucleicacid when complexed with a guide RNA) includes a substitution of the Aspat position 413 of Cas12J_3339380 (FIG. 6D), or a corresponding positionin another Cas12J, with an amino acid other than Asp. As an example, anamino acid substitution that results in a dCas12J polypeptide (i.e., aCas12J polypeptide that binds, but does not cleave, a target nucleicacid when complexed with a guide RNA) includes a D413A substitution atposition 413 of Cas12J_3339380 (FIG. 6D), or a corresponding position inanother Cas12J.

An amino acid substitution that results in a dCas12J polypeptide (i.e.,a Cas12J polypeptide that binds, but does not cleave, a target nucleicacid when complexed with a guide RNA) includes a substitution of the Aspat position 371 of Cas12J_1947455 (FIG. 6A), or a corresponding positionin another Cas12J, with an amino acid other than Asp. As an example, anamino acid substitution that results in a dCas12J polypeptide (i.e., aCas12J polypeptide that binds, but does not cleave, a target nucleicacid when complexed with a guide RNA) includes a D371A substitution atposition 371 of Cas12J_1947455 (FIG. 6A), or a corresponding position inanother Cas12J.

An amino acid substitution that results in a dCas12J polypeptide (i.e.,a Cas12J polypeptide that binds, but does not cleave, a target nucleicacid when complexed with a guide RNA) includes a substitution of the Aspat position 394 of Cas12J_2071242 (FIG. 6B), or a corresponding positionin another Cas12J, with an amino acid other than Asp. As an example, anamino acid substitution that results in a dCas12J polypeptide (i.e., aCas12J polypeptide that binds, but does not cleave, a target nucleicacid when complexed with a guide RNA) includes a D394A substitution atposition 394 of Cas12J_2071242 (FIG. 6B), or a corresponding position inanother Cas12J.

Amino acid positions corresponding to the Asp at position 413 ofCas12J_3339380 (FIG. 6D) (CasΦ-3), the Asp at position 371 ofCas12J_1947455 (FIG. 6A) (CasΦ-1), and the Asp at position 394 ofCas12J_2071242 (FIG. 6B) (CasΦ-2), can be readily determined by, e.g.,aligning the amino acid sequences of the Cas12J polypeptides depicted inFIG. 6A-6R. For example, amino acid positions corresponding to the Aspat position 413 of Cas12J_3339380 (FIG. 6D), the Asp at position 371 ofCas12J_1947455 (FIG. 6A), and the Asp at position 394 of Cas12J_2071242(FIG. 6B), are depicted in FIG. 9. For example, the Asp in Ruv-CI that,when substituted with an amino acid other than Asp, can in a dCas12Jpolypeptide includes:

1) Asp-371 of the Cas12J polypeptide designated “Cas12J_1947455” (or“Cas12J_1947455_11” in FIG. 9) and depicted in FIG. 6A (“CasΦ-1”);

2) Asp-394 of the Cas12J polypeptide designated “Cas12J_2071242” anddepicted in FIG. 6B (“CasΦ-2”);

3) Asp-413 of the Cas12J polypeptide designated “Cas12J_3339380 (or“Cas12J_3339380_12” in FIG. 9) and depicted in FIG. 6D (“CasΦ-3”);

4) Asp-419 of the Cas12J polypeptide designated “Cas12J_3877103_16” anddepicted in FIG. 6Q (“CasΦ-4”);

5) Asp-416 of the Cas12J polypeptide designated “Cas12J_10000002_47” or“Cas12J_1000002_112” and depicted in FIG. 6G (“CasΦ-5”);

6) Asp-384 of the Cas12J polypeptide designated “Cas12J_10100763_4” anddepicted in FIG. 6H (“CasΦ-6”);

7) Asp-423 of the Cas12J polypeptide designated “Cas12J_1000007_143” or“Cas12J_1000001_267” and depicted in FIG. 6P (“CasΦ-7”);

8) Asp-369 of the Cas12J polypeptide designated “Cas12J_10000286_53” anddepicted in FIG. 6L (or “Cas12J_10000506_8” and depicted in FIG. 6O)(“CasΦ-8”);

9) Asp-426 of the Cas12J polypeptide designated “Cas12J_10001283_7” anddepicted in FIG. 6M (“CasΦ-9”);

10) Asp-464 of the Cas12J polypeptide designated “Cas12J_10037042_3” anddepicted in FIG. 6E (“CasΦ-10”).

Variants—Fusion Cas12J Polypeptides

As noted above, in some cases, a Cas12J protein (in some cases a Cas12Jprotein with wild type cleavage activity and in some cases a variantCas12J with reduced cleavage activity, e.g., a dCas12J or a nickaseCas12J) is fused (conjugated) to a heterologous polypeptide (i.e., oneor more heterologous polypeptides) that has an activity of interest(e.g., a catalytic activity of interest) to form a fusion protein. Aheterologous polypeptide to which a Cas12J protein can be fused isreferred to herein as a “fusion partner.”

In some cases, the fusion partner can modulate transcription (e.g.,inhibit transcription, increase transcription) of a target DNA. Forexample, in some cases the fusion partner is a protein (or a domain froma protein) that inhibits transcription (e.g., a transcriptionalrepressor, a protein that functions via recruitment of transcriptioninhibitor proteins, modification of target DNA such as methylation,recruitment of a DNA modifier, modulation of histones associated withtarget DNA, recruitment of a histone modifier such as those that modifyacetylation and/or methylation of histones, and the like). In somecases, the fusion partner is a protein (or a domain from a protein) thatincreases transcription (e.g., a transcription activator, a protein thatacts via recruitment of transcription activator proteins, modificationof target DNA such as demethylation, recruitment of a DNA modifier,modulation of histones associated with target DNA, recruitment of ahistone modifier such as those that modify acetylation and/ormethylation of histones, and the like). In some cases, the fusionpartner is a reverse transcriptase. In some cases, the fusion partner isa base editor. In some cases, the fusion partner is a deaminase.

In some cases, a fusion Cas12J protein includes a heterologouspolypeptide that has enzymatic activity that modifies a target nucleicacid (e.g., nuclease activity, methyltransferase activity, demethylaseactivity, DNA repair activity, DNA damage activity, deaminationactivity, dismutase activity, alkylation activity, depurinationactivity, oxidation activity, pyrimidine dimer forming activity,integrase activity, transposase activity, recombinase activity,polymerase activity, ligase activity, helicase activity, photolyaseactivity, or glycosylase activity).

In some cases, a fusion Cas12J protein includes a heterologouspolypeptide that has enzymatic activity that modifies a polypeptide(e.g., a histone) associated with a target nucleic acid (e.g.,methyltransferase activity, demethylase activity, acetyltransferaseactivity, deacetylase activity, kinase activity, phosphatase activity,ubiquitin ligase activity, deubiquitinating activity, adenylationactivity, deadenylation activity, SUMOylating activity, deSUMOylatingactivity, ribosylation activity, deribosylation activity, myristoylationactivity or demyristoylation activity).

Examples of proteins (or fragments thereof) that can be used in increasetranscription include but are not limited to: transcriptional activatorssuch as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), andactivation domain of EDLL and/or TAL activation domain (e.g., foractivity in plants); histone lysine methyltransferases such as SET1A,SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysinedemethylases such as JHDM2a/b, UTX, JMJD3, and the like; histoneacetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP,MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNAdemethylases such as Ten-Eleven Translocation (TET) dioxygenase 1(TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.

Examples of proteins (or fragments thereof) that can be used in decreasetranscription include but are not limited to: transcriptional repressorssuch as the Krüppel associated box (KRAB or SKD); KOX1 repressiondomain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain(ERD), the SRDX repression domain (e.g., for repression in plants), andthe like; histone lysine methyltransferases such as Pr-SET7/8,SUV4-20H1, RIZ1, and the like; histone lysine demethylases such asJMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2,JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, and the like; histone lysinedeacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7,HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as HhaIDNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNAmethyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI,DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and peripheryrecruitment elements such as Lamin A, Lamin B, and the like.

In some cases, the fusion partner has enzymatic activity that modifiesthe target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples ofenzymatic activity that can be provided by the fusion partner includebut are not limited to: nuclease activity such as that provided by arestriction enzyme (e.g., FokI nuclease), methyltransferase activitysuch as that provided by a methyltransferase (e.g., HhaI DNAm5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNAmethyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI,DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylaseactivity such as that provided by a demethylase (e.g., Ten-ElevenTranslocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1,and the like), DNA repair activity, DNA damage activity, deaminationactivity such as that provided by a deaminase (e.g., a cytosinedeaminase enzyme such as rat APOBEC1), dismutase activity, alkylationactivity, depurination activity, oxidation activity, pyrimidine dimerforming activity, integrase activity such as that provided by anintegrase and/or resolvase (e.g., Gin invertase such as the hyperactivemutant of the Gin invertase, GinH106Y; human immunodeficiency virus type1 integrase (IN); Tn3 resolvase; and the like), transposase activity,recombinase activity such as that provided by a recombinase (e.g.,catalytic domain of Gin recombinase), polymerase activity, ligaseactivity, helicase activity, photolyase activity, and glycosylaseactivity).

In some cases, the fusion partner has enzymatic activity that modifies aprotein associated with the target nucleic acid (e.g., ssRNA, dsRNA,ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA bindingprotein, and the like). Examples of enzymatic activity (that modifies aprotein associated with a target nucleic acid) that can be provided bythe fusion partner include but are not limited to: methyltransferaseactivity such as that provided by a histone methyltransferase (HMT)(e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known asKMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also knownas KMT1C and EHMT2), SUV39H2, ESET/SETDB1, and the like, SET1A, SET1B,MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1),demethylase activity such as that provided by a histone demethylase(e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b,JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2,JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, UTX, JMJD3, and the like),acetyltransferase activity such as that provided by a histone acetylasetransferase (e.g., catalytic core/fragment of the humanacetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60/PLIP, MOZ/MYST3,MORF/MYST4, HBO1/MYST2, HMOF/MYST1, SRC1, ACTR, P160, CLOCK, and thelike), deacetylase activity such as that provided by a histonedeacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7,HDAC9, SIRT1, SIRT2, HDAC11, and the like), kinase activity, phosphataseactivity, ubiquitin ligase activity, deubiquitinating activity,adenylation activity, deadenylation activity, SUMOylating activity,deSUMOylating activity, ribosylation activity, deribosylation activity,myristoylation activity, and demyristoylation activity.

Additional examples of a suitable fusion partners are dihydrofolatereductase (DHFR) destabilization domain (e.g., to generate a chemicallycontrollable fusion Cas12J protein), and a chloroplast transit peptide.Suitable chloroplast transit peptides include, but are not limited to:

(SEQ ID NO: 25) MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGRVKCMQVWPPIGKKKFETLSYLPPLTRDSRA; (SEQ ID NO: 26)MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSN GGRVKS;(SEQ ID NO: 27) MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQVWPPIEKKKFETLSYLPDLTDSGGRVNC; (SEQ ID NO: 28)MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIGSELRPLKVMSSVSTAC; (SEQ ID NO: 29)MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIGSELRPLKVMSSVSTAC; (SEQ ID NO: 30)MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVLKKDSIFMQLFCSFRISASVATAC; (SEQ ID NO: 31)MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTVGASAAP KQSRKPHRFDRRCLSMVV;(SEQ ID NO: 32) MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQQRSVQRGSRRFPSVVVC; (SEQ ID NO: 33)MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASN GGRVQC;(SEQ ID NO: 34) MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVKCSAAVTPQASPVISRSAAAA; and (SEQ ID NO: 35)MGAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCASSWNSTINGAAATTNGASAASS.

In some case, a Cas12J fusion polypeptide of the present disclosurecomprises: a) a Cas12J polypeptide of the present disclosure; and b) achloroplast transit peptide. Thus, for example, a Cas12Jpolypeptide/guide RNA complex can be targeted to the chloroplast. Insome cases, this targeting may be achieved by the presence of anN-terminal extension, called a chloroplast transit peptide (CTP) orplastid transit peptide. Chromosomal transgenes from bacterial sourcesmust have a sequence encoding a CTP sequence fused to a sequenceencoding an expressed polypeptide if the expressed polypeptide is to becompartmentalized in the plant plastid (e.g. chloroplast). Accordingly,localization of an exogenous polypeptide to a chloroplast is often 1accomplished by means of operably linking a polynucleotide sequenceencoding a CTP sequence to the 5′ region of a polynucleotide encodingthe exogenous polypeptide. The CTP is removed in a processing stepduring translocation into the plastid. Processing efficiency may,however, be affected by the amino acid sequence of the CTP and nearbysequences at the amino terminus (NH₂ terminus) of the peptide. Otheroptions for targeting to the chloroplast which have been described arethe maize cab-m7 signal sequence (U.S. Pat. No. 7,022,896, WO 97/41228)a pea glutathione reductase signal sequence (WO 97/41228) and the CTPdescribed in US2009029861.

In some cases, a Cas12J fusion polypeptide of the present disclosure cancomprise: a) a Cas12J polypeptide of the present disclosure; and b) anendosomal escape peptide. In some cases, an endosomal escape polypeptidecomprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 36),wherein each X is independently selected from lysine, histidine, andarginine. In some cases, an endosomal escape polypeptide comprises theamino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 37).

For examples of some of the above fusion partners (and more) used in thecontext of fusions with Cas9, Zinc Finger, and/or TALE proteins (forsite specific target nucleic modification, modulation of transcription,and/or target protein modification, e.g., histone modification), see,e.g.: Nomura et al, J Am Chem Soc. 2007 Jul. 18; 129(28):8676-7;Rivenbark et al., Epigenetics. 2012 April; 7(4):350-60; Nucleic AcidsRes. 2016 Jul. 8; 44(12):5615-28; Gilbert et al., Cell. 2013 Jul. 18;154(2):442-51; Kearns et al., Nat Methods. 2015 May; 12(5):401-3;Mendenhall et al., Nat Biotechnol. 2013 December; 31(12):1133-6; Hiltonet al., Nat Biotechnol. 2015 May; 33(5):510-7; Gordley et al., Proc NatlAcad Sci USA. 2009 Mar. 31; 106(13):5053-8; Akopian et al., Proc NatlAcad Sci USA. 2003 Jul. 22; 100(15):8688-91; Tan et., al., J Virol. 2006February; 80(4):1939-48; Tan et al., Proc Natl Acad Sci USA. 2003 Oct.14; 100(21):11997-2002; Papworth et al., Proc Natl Acad Sci USA. 2003Feb. 18; 100(4):1621-6; Sanjana et al., Nat Protoc. 2012 Jan. 5;7(1):171-92; Beerli et al., Proc Natl Acad Sci USA. 1998 Dec. 8;95(25):14628-33; Snowden et al., Curr Biol. 2002 Dec. 23;12(24):2159-66; Xu et.al., Xu et al., Cell Discov. 2016 May 3; 2:16009;Komor et al., Nature. 2016 Apr. 20; 533(7603):420-4; Chaikind et al.,Nucleic Acids Res. 2016 Aug. 11; Choudhury at. al., Oncotarget. 2016Jun. 23; Du et al., Cold Spring Harb Protoc. 2016 Jan. 4; Pham et al.,Methods Mol Biol. 2016; 1358:43-57; Balboa et al., Stem Cell Reports.2015 Sep. 8; 5(3):448-59; Hara et al., Sci Rep. 2015 Jun. 9; 5:11221;Piatek et al., Plant Biotechnol J. 2015 May; 13(4):578-89; Hu et al.,Nucleic Acids Res. 2014 April; 42(7):4375-90; Cheng et al., Cell Res.2013 October; 23(10):1163-71; and Maeder et al., Nat Methods. 2013October; 10(10):977-9.

Additional suitable heterologous polypeptides include, but are notlimited to, a polypeptide that directly and/or indirectly provides forincreased or decreased transcription and/or translation of a targetnucleic acid (e.g., a transcription activator or a fragment thereof, aprotein or fragment thereof that recruits a transcription activator, asmall molecule/drug-responsive transcription and/or translationregulator, a translation-regulating protein, etc.). Non-limitingexamples of heterologous polypeptides to accomplish increased ordecreased transcription include transcription activator andtranscription repressor domains. In some such cases, a fusion Cas12Jpolypeptide is targeted by the guide nucleic acid (guide RNA) to aspecific location (i.e., sequence) in the target nucleic acid and exertslocus-specific regulation such as blocking RNA polymerase binding to apromoter (which selectively inhibits transcription activator function),and/or modifying the local chromatin status (e.g., when a fusionsequence is used that modifies the target nucleic acid or modifies apolypeptide associated with the target nucleic acid). In some cases, thechanges are transient (e.g., transcription repression or activation). Insome cases, the changes are inheritable (e.g., when epigeneticmodifications are made to the target nucleic acid or to proteinsassociated with the target nucleic acid, e.g., nucleosomal histones).

Non-limiting examples of heterologous polypeptides for use whentargeting ssRNA target nucleic acids include (but are not limited to):splicing factors (e.g., RS domains); protein translation components(e.g., translation initiation, elongation, and/or release factors; e.g.,eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g.,adenosine deaminase acting on RNA (ADAR), including A to I and/or C to Uediting enzymes); helicases; RNA-binding proteins; and the like. It isunderstood that a heterologous polypeptide can include the entireprotein or in some cases can include a fragment of the protein (e.g., afunctional domain).

The heterologous polypeptide of a subject fusion Cas12J polypeptide canbe any domain capable of interacting with ssRNA (which, for the purposesof this disclosure, includes intramolecular and/or intermolecularsecondary structures, e.g., double-stranded RNA duplexes such ashairpins, stem-loops, etc.), whether transiently or irreversibly,directly or indirectly, including but not limited to an effector domainselected from the group comprising; Endonucleases (for example RNaseIII, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains fromproteins such as SMG5 and SMG6); proteins and protein domainsresponsible for stimulating RNA cleavage (for example CPSF, CstF, CFImand CFIIm); Exonucleases (for example XRN-1 or Exonuclease T);Deadenylases (for example HNT3); proteins and protein domainsresponsible for nonsense mediated RNA decay (for example UPF1, UPF2,UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); proteins and proteindomains responsible for stabilizing RNA (for example PABP); proteins andprotein domains responsible for repressing translation (for example Ago2and Ago4); proteins and protein domains responsible for stimulatingtranslation (for example Staufen); proteins and protein domainsresponsible for (e.g., capable of) modulating translation (e.g.,translation factors such as initiation factors, elongation factors,release factors, etc., e.g., eIF4G); proteins and protein domainsresponsible for polyadenylation of RNA (for example PAP1, GLD-2, andStar-PAP); proteins and protein domains responsible forpolyuridinylation of RNA (for example CI D1 and terminal uridylatetransferase); proteins and protein domains responsible for RNAlocalization (for example from IMP1, ZBP1, She2p, She3p, andBicaudal-D); proteins and protein domains responsible for nuclearretention of RNA (for example Rrp6); proteins and protein domainsresponsible for nuclear export of RNA (for example TAP, NXF1, THO, TREX,REF, and Aly); proteins and protein domains responsible for repressionof RNA splicing (for example PTB, Sam68, and hnRNP A1); proteins andprotein domains responsible for stimulation of RNA splicing (for exampleSerine/Arginine-rich (SR) domains); proteins and protein domainsresponsible for reducing the efficiency of transcription (for exampleFUS (TLS)); and proteins and protein domains responsible for stimulatingtranscription (for example CDK7 and HIV Tat). Alternatively, theeffector domain may be selected from the group comprising Endonucleases;proteins and protein domains capable of stimulating RNA cleavage;Exonucleases; Deadenylases; proteins and protein domains having nonsensemediated RNA decay activity; proteins and protein domains capable ofstabilizing RNA; proteins and protein domains capable of repressingtranslation; proteins and protein domains capable of stimulatingtranslation; proteins and protein domains capable of modulatingtranslation (e.g., translation factors such as initiation factors,elongation factors, release factors, etc., e.g., eIF4G); proteins andprotein domains capable of polyadenylation of RNA; proteins and proteindomains capable of polyuridinylation of RNA; proteins and proteindomains having RNA localization activity; proteins and protein domainscapable of nuclear retention of RNA; proteins and protein domains havingRNA nuclear export activity; proteins and protein domains capable ofrepression of RNA splicing; proteins and protein domains capable ofstimulation of RNA splicing; proteins and protein domains capable ofreducing the efficiency of transcription; and proteins and proteindomains capable of stimulating transcription. Another suitableheterologous polypeptide is a PUF RNA-binding domain, which is describedin more detail in WO2012068627, which is hereby incorporated byreference in its entirety.

Some RNA splicing factors that can be used (in whole or as fragmentsthereof) as heterologous polypeptides for a fusion Cas12J polypeptidehave modular organization, with separate sequence-specific RNA bindingmodules and splicing effector domains. For example, members of theSerine/Arginine-rich (SR) protein family contain N-terminal RNArecognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs)in pre-mRNAs and C-terminal RS domains that promote exon inclusion. Asanother example, the hnRNP protein hnRNP A1 binds to exonic splicingsilencers (ESSs) through its RRM domains and inhibits exon inclusionthrough a C-terminal Glycine-rich domain. Some splicing factors canregulate alternative use of splice site (ss) by binding to regulatorysequences between the two alternative sites. For example, ASF/SF2 canrecognize ESEs and promote the use of intron proximal sites, whereashnRNP A1 can bind to ESSs and shift splicing towards the use of introndistal sites. One application for such factors is to generate ESFs thatmodulate alternative splicing of endogenous genes, particularly diseaseassociated genes. For example, Bcl-x pre-mRNA produces two splicingisoforms with two alternative 5′ splice sites to encode proteins ofopposite functions. The long splicing isoform Bcl-xL is a potentapoptosis inhibitor expressed in long-lived postmitotic cells and isup-regulated in many cancer cells, protecting cells against apoptoticsignals. The short isoform Bcl-xS is a pro-apoptotic isoform andexpressed at high levels in cells with a high turnover rate (e.g.,developing lymphocytes). The ratio of the two Bcl-x splicing isoforms isregulated by multiple c{acute over (ω)}-elements that are located ineither the core exon region or the exon extension region (i.e., betweenthe two alternative 5′ splice sites). For more examples, seeWO2010075303, which is hereby incorporated by reference in its entirety.

Further suitable fusion partners include, but are not limited to,proteins (or fragments thereof) that are boundary elements (e.g., CTCF),proteins and fragments thereof that provide periphery recruitment (e.g.,Lamin A, Lamin B, etc.), protein docking elements (e.g., FKBP/FRB,Pil1/Aby1, etc.).

Nucleases

In some cases, a subject fusion Cas12J polypeptide comprises: i) aCas12J polypeptide of the present disclosure; and ii) a heterologouspolypeptide (a “fusion partner”), where the heterologous polypeptide isa nuclease. Suitable nucleases include, but are not limited to, a homingnuclease polypeptide; a FokI polypeptide; a transcription activator-likeeffector nuclease (TALEN) polypeptide; a MegaTAL polypeptide; ameganuclease polypeptide; a zinc finger nuclease (ZFN); an ARCUSnuclease; and the like. The meganuclease can be engineered from anLADLIDADG homing endonuclease (LHE). A megaTAL polypeptide can comprisea TALE DNA binding domain and an engineered meganuclease. See, e.g., WO2004/067736 (homing endonuclease); Urnov et al. (2005) Nature 435:646(ZFN); Mussolino et al. (2011) Nude. Acids Res. 39:9283 (TALE nuclease);Boissel et al. (2013) Nucl. Acids Res. 42:2591 (MegaTAL).

Reverse Transcriptases

In some cases, a subject fusion Cas12J polypeptide comprises: i) aCas12J polypeptide of the present disclosure; and ii) a heterologouspolypeptide (a “fusion partner”), where the heterologous polypeptide isa reverse transcriptase polypeptide. In some cases, the Cas12Jpolypeptide is catalytically inactive. Suitable reverse transcriptasesinclude, e.g., a murine leukemia virus reverse transcriptase; a Roussarcoma virus reverse transcriptase; a human immunodeficiency virus typeI reverse transcriptase; a Moloney murine leukemia virus reversetranscriptase; and the like.

Base Editors

In some cases, a Cas12J fusion polypeptide of the present disclosurecomprises: i) a Cas12J polypeptide of the present disclosure; and ii) aheterologous polypeptide (a “fusion partner”), where the heterologouspolypeptide is a base editor. Suitable base editors include, e.g., anadenosine deaminase; a cytidine deaminase (e.g., an activation-inducedcytidine deaminase (AID)); APOBEC3G; and the like); and the like.

A suitable adenosine deaminase is any enzyme that is capable ofdeaminating adenosine in DNA. In some cases, the deaminase is a TadAdeaminase.

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 38) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRR QEIKAQKKAQSSTD

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 39) MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD.

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing Staphylococcus aureus TadA amino acid sequence:

(SEQ ID NO: 40) MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFK NLRANKKSTN

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing Bacillus subtilis TadA amino acid sequence:

(SEQ ID NO: 41) MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRK KKKAARKNLSE

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing Salmonella typhimurium TadA:

(SEQ ID NO: 42) MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing Shewanella putrefaciens TadA amino acid sequence:

(SEQ ID NO: 43) MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEK KALKLAQRAQQGIE

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing Haemophilus influenzae F3031 TadA amino acid sequence:

(SEQ ID NO: 44) MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing Caulobacter crescentus TadA amino acid sequence:

(SEQ ID NO: 45) MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLR GFFRARRKAKI

In some cases, a suitable adenosine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing Geobacter sulfurreducens TadA amino acid sequence:

(SEQ ID NO: 46) MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

Cytidine deaminases suitable for inclusion in a CRISPR/Cas effectorpolypeptide fusion polypeptide include any enzyme that is capable ofdeaminating cytidine in DNA.

In some cases, the cytidine deaminase is a deaminase from theapolipoprotein B mRNA-editing complex (APOBEC) family of deaminases. Insome cases, the APOBEC family deaminase is selected from the groupconsisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase,APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3Fdeaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases,the cytidine deaminase is an activation induced deaminase (AID).

In some cases, a suitable cytidine deaminase comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 47) MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL

In some cases, a suitable cytidine deaminase is an AID and comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 48) MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSATSFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTWFTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRKAEPEGLRRLH RAGVQIAIMT FKENHERTFK AWEGLHENSVRLSRQLRRIL LPLYEVDDLR DAFRTLGL.

In some cases, a suitable cytidine deaminase is an AID and comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 47) MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSATSFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTWFTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRKAEPEGLRRLH RAGVQIAIMT FKDYFYCWNT FVENHERTFKAWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL.Transcription Factors

In some cases, a Cas12J fusion polypeptide of the present disclosurecomprises: i) a Cas12J polypeptide of the present disclosure; and ii) aheterologous polypeptide (a “fusion partner”), where the heterologouspolypeptide is a transcription factor. A transcription factor caninclude: i) a DNA binding domain; and ii) a transcription activator. Atranscription factor can include: i) a DNA binding domain; and ii) atranscription repressor. Suitable transcription factors includepolypeptides that include a transcription activator or a transcriptionrepressor domain (e.g., the Kruppel associated box (KRAB or SKD); theMad mSIN3 interaction domain (SID); the ERF repressor domain (ERD),etc.); zinc-finger-based artificial transcription factors (see, e.g.,Sera (2009) Adv. Drug Deliv. 61:513); TALE-based artificialtranscription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics14:781); and the like. In some cases, the transcription factor comprisesa VP64 polypeptide (transcriptional activation). In some cases, thetranscription factor comprises a Kruppel-associated box (KRAB)polypeptide (transcriptional repression). In some cases, thetranscription factor comprises a Mad mSIN3 interaction domain (SID)polypeptide (transcriptional repression). In some cases, thetranscription factor comprises an ERF repressor domain (ERD) polypeptide(transcriptional repression). For example, in some cases, thetranscription factor is a transcriptional activator, where thetranscriptional activator is GAL4-VP16.

Recombinases

In some cases, a Cas12J fusion polypeptide of the present disclosurecomprises: i) a Cas12J polypeptide of the present disclosure; and ii) aheterologous polypeptide (a “fusion partner”), where the heterologouspolypeptide is a recombinase. Suitable recombinases include, e.g., a Crerecombinase; a Hin recombinase; a Tre recombinase; a FLP recombinase;and the like.

Examples of various additional suitable heterologous polypeptide (orfragments thereof) for a subject fusion Cas12J polypeptide include, butare not limited to, those described in the following applications (whichpublications are related to other CRISPR endonucleases such as Cas9, butthe described fusion partners can also be used with Cas12J instead): PCTpatent applications: WO2010075303, WO2012068627, and WO2013155555, andcan be found, for example, in U.S. patents and patent applications: U.S.Pat. Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445;8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753;20140179006; 20140179770; 20140186843; 20140186919; 20140186958;20140189896; 20140227787; 20140234972; 20140242664; 20140242699;20140242700; 20140242702; 20140248702; 20140256046; 20140273037;20140273226; 20140273230; 20140273231; 20140273232; 20140273233;20140273234; 20140273235; 20140287938; 20140295556; 20140295557;20140298547; 20140304853; 20140309487; 20140310828; 20140310830;20140315985; 20140335063; 20140335620; 20140342456; 20140342457;20140342458; 20140349400; 20140349405; 20140356867; 20140356956;20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and20140377868; all of which are hereby incorporated by reference in theirentirety.

In some cases, a heterologous polypeptide (a fusion partner) providesfor subcellular localization, i.e., the heterologous polypeptidecontains a subcellular localization sequence (e.g., a nuclearlocalization signal (NLS) for targeting to the nucleus, a sequence tokeep the fusion protein out of the nucleus, e.g., a nuclear exportsequence (NES), a sequence to keep the fusion protein retained in thecytoplasm, a mitochondrial localization signal for targeting to themitochondria, a chloroplast localization signal for targeting to achloroplast, an ER retention signal, and the like). In some cases, aCas12J fusion polypeptide does not include an NLS so that the protein isnot targeted to the nucleus (which can be advantageous, e.g., when thetarget nucleic acid is an RNA that is present in the cytosol). In somecases, the heterologous polypeptide can provide a tag (i.e., theheterologous polypeptide is a detectable label) for ease of trackingand/or purification (e.g., a fluorescent protein, e.g., greenfluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry,tdTomato, and the like; a histidine tag, e.g., a 6×His tag; ahemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).

In some cases, a Cas12J protein (e.g., a wild type Cas12J protein, avariant Cas12J protein, a fusion Cas12J protein, a dCas12J protein, andthe like) includes (is fused to) a nuclear localization signal (NLS)(e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or moreNLSs). Thus, in some cases, a Cas12J polypeptide includes one or moreNLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In somecases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or moreNLSs) are positioned at or near (e.g., within 50 amino acids of) theN-terminus and/or the C-terminus. In some cases, one or more NLSs (2 ormore, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near(e.g., within 50 amino acids of) the N-terminus. In some cases, one ormore NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) arepositioned at or near (e.g., within 50 amino acids of) the C-terminus.In some cases, one or more NLSs (3 or more, 4 or more, or 5 or moreNLSs) are positioned at or near (e.g., within 50 amino acids of) boththe N-terminus and the C-terminus. In some cases, an NLS is positionedat the N-terminus and an NLS is positioned at the C-terminus.

In some cases, a Cas12J protein (e.g., a wild type Cas12J protein, avariant Cas12J protein, a fusion Cas12J protein, a dCas12J protein, andthe like) includes (is fused to) between 1 and 10 NLSs (e.g., 1-9, 1-8,1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5 NLSs). In some cases, aCas12J protein (e.g., a wild type Cas12J protein, a variant Cas12Jprotein, a fusion Cas12J protein, a dCas12J protein, and the like)includes (is fused to) between 2 and 5 NLSs (e.g., 2-4, or 2-3 NLSs).

Non-limiting examples of NLSs include an NLS sequence derived from: theNLS of the SV40 virus large T-antigen, having the amino acid sequencePKKKRKV (SEQ ID NO: 49); the NLS from nucleoplasmin (e.g., thenucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ IDNO: 50)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ IDNO: 51) or RQRRNELKRSP (SEQ ID NO: 52); the hRNPA1 M9 NLS having thesequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 53); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 54) ofthe IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:55) and PPKKARED (SEQ ID NO: 98) of the myoma T protein; the sequencePQPKKKPL (SEQ ID NO: 56) of human p53; the sequence SALIKKKKKMAP (SEQ IDNO: 57) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 58) andPKQKKRK (SEQ ID NO: 59) of the influenza virus NS1; the sequenceRKLKKKIKKL (SEQ ID NO: 60) of the Hepatitis virus delta antigen; thesequence REKKKFLKRR (SEQ ID NO: 61) of the mouse M×1 protein; thesequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 62) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 63) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, NLS (or multiple NLSs) are of sufficient strength to driveaccumulation of the Cas12J protein in a detectable amount in the nucleusof a eukaryotic cell. Detection of accumulation in the nucleus may beperformed by any suitable technique. For example, a detectable markermay be fused to the Cas12J protein such that location within a cell maybe visualized. Cell nuclei may also be isolated from cells, the contentsof which may then be analyzed by any suitable process for detectingprotein, such as immunohistochemistry, Western blot, or enzyme activityassay. Accumulation in the nucleus may also be determined indirectly.

In some cases, a Cas12J fusion polypeptide includes a “ProteinTransduction Domain” or PTD (also known as a CPP—cell penetratingpeptide), which refers to a polypeptide, polynucleotide, carbohydrate,or organic or inorganic compound that facilitates traversing a lipidbilayer, micelle, cell membrane, organelle membrane, or vesiclemembrane. A PTD attached to another molecule, which can range from asmall polar molecule to a large macromolecule and/or a nanoparticle,facilitates the molecule traversing a membrane, for example going fromextracellular space to intracellular space, or cytosol to within anorganelle. In some embodiments, a PTD is covalently linked to the aminoterminus a polypeptide (e.g., linked to a wild type Cas12J to generate afusion protein, or linked to a variant Cas12J protein such as a dCas12J,nickase Cas12J, or fusion Cas12J protein, to generate a fusion protein).In some embodiments, a PTD is covalently linked to the carboxyl terminusof a polypeptide (e.g., linked to a wild type Cas12J to generate afusion protein, or linked to a variant Cas12J protein such as a dCas12J,nickase Cas12J, or fusion Cas12J protein to generate a fusion protein).In some cases, the PTD is inserted internally in the Cas12J fusionpolypeptide (i.e., is not at the N- or C-terminus of the Cas12J fusionpolypeptide) at a suitable insertion site. In some cases, a subjectCas12J fusion polypeptide includes (is conjugated to, is fused to) oneor more PTDs (e.g., two or more, three or more, four or more PTDs). Insome cases, a PTD includes a nuclear localization signal (NLS) (e.g, insome cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, insome cases, a Cas12J fusion polypeptide includes one or more NLSs (e.g.,2 or more, 3 or more, 4 or more, or 5 or more NLSs). In someembodiments, a PTD is covalently linked to a nucleic acid (e.g., aCas12J guide nucleic acid, a polynucleotide encoding a Cas12J guidenucleic acid, a polynucleotide encoding a Cas12J fusion polypeptide, adonor polynucleotide, etc.). Examples of PTDs include but are notlimited to a minimal undecapeptide protein transduction domain(corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR;SEQ ID NO: 64); a polyarginine sequence comprising a number of argininessufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10,or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer GeneTher. 9(6):489-96); an Drosophila Antennapedia protein transductiondomain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncatedhuman calcitonin peptide (Trehin et al. (2004) Pharm. Research21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci.USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: 65); TransportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 66);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 67); and RQIKIWFQNRRMKWKK(SEQ ID NO: 68). Exemplary PTDs include but are not limited to,YGRKKRRQRRR (SEQ ID NO: 64), RKKRRQRRR (SEQ ID NO: 70); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;Exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following: YGRKKRRQRRR (SEQ ID NO: 64); RKKRRQRR (SEQ IDNO: 70); YARAAARQARA (SEQ ID NO: 71); THRLPRRRRRR (SEQ ID NO: 72); andGGRRARRRRRR (SEQ ID NO: 73). In some embodiments, the PTD is anactivatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”)connected via a cleavable linker to a matching polyanion (e.g., Glu9 or“E9”), which reduces the net charge to nearly zero and thereby inhibitsadhesion and uptake into cells. Upon cleavage of the linker, thepolyanion is released, locally unmasking the polyarginine and itsinherent adhesiveness, thus “activating” the ACPP to traverse themembrane.

Linkers (e.g., for Fusion Partners)

In some embodiments, a subject Cas12J protein can fused to a fusionpartner via a linker polypeptide (e.g., one or more linkerpolypeptides). The linker polypeptide may have any of a variety of aminoacid sequences. Proteins can be joined by a spacer peptide, generally ofa flexible nature, although other chemical linkages are not excluded.Suitable linkers include polypeptides of between 4 amino acids and 40amino acids in length, or between 4 amino acids and 25 amino acids inlength. These linkers can be produced by using synthetic,linker-encoding oligonucleotides to couple the proteins, or can beencoded by a nucleic acid sequence encoding the fusion protein. Peptidelinkers with a degree of flexibility can be used. The linking peptidesmay have virtually any amino acid sequence, bearing in mind that thepreferred linkers will have a sequence that results in a generallyflexible peptide. The use of small amino acids, such as glycine andalanine, are of use in creating a flexible peptide. The creation of suchsequences is routine to those of skill in the art. A variety ofdifferent linkers are commercially available and are considered suitablefor use.

Examples of linker polypeptides include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n)(SEQ ID NO: 74), GGSGGS_(n) (SEQ ID NO: 75), and GGGS_(n) (SEQ ID NO:76), where n is an integer of at least one), glycine-alanine polymers,alanine-serine polymers. Exemplary linkers can comprise amino acidsequences including, but not limited to, GGSG (SEQ ID NO: 77), GGSGG(SEQ ID NO: 78), GSGSG (SEQ ID NO: 79), GSGGG (SEQ ID NO: 80), GGGSG(SEQ ID NO: 81), GSSSG (SEQ ID NO: 82), and the like. The ordinarilyskilled artisan will recognize that design of a peptide conjugated toany desired element can include linkers that are all or partiallyflexible, such that the linker can include a flexible linker as well asone or more portions that confer less flexible structure.

Detectable Labels

In some cases, a Cas12J polypeptide of the present disclosure comprisesa detectable label. Suitable detectable labels and/or moieties that canprovide a detectable signal can include, but are not limited to, anenzyme, a radioisotope, a member of a specific binding pair; afluorophore; a fluorescent protein; a quantum dot; and the like.

Suitable fluorescent proteins include, but are not limited to, greenfluorescent protein (GFP) or variants thereof, blue fluorescent variantof GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescentvariant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhancedYFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine,GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP),destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet,mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2,t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP,Kaede protein and kindling protein, Phycobiliproteins andPhycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrinand Allophycocyanin. Other examples of fluorescent proteins includemHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry,mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat.Methods 2:905-909), and the like. Any of a variety of fluorescent andcolored proteins from Anthozoan species, as described in, e.g., Matz etal. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

Suitable enzymes include, but are not limited to, horse radishperoxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL),glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase,glucose oxidase (GO), and the like.

Protospacer Adjacent Motif (PAM)

A Cas12J protein binds to target DNA at a target sequence defined by theregion of complementarity between the DNA-targeting RNA and the targetDNA. As is the case for many CRISPR endonucleases, site-specific binding(and/or cleavage) of a double stranded target DNA occurs at locationsdetermined by both (i) base-pairing complementarity between the guideRNA and the target DNA; and (ii) a short motif [referred to as theprotospacer adjacent motif (PAM)] in the target DNA.

In some embodiments, the PAM for a Cas12J protein is immediately 5′ ofthe target sequence of the non-complementary strand of the target DNA(the complementary strand: (i) hybridizes to the guide sequence of theguide RNA, while the non-complementary strand does not directlyhybridize with the guide RNA; and (ii) is the reverse complement of thenon-complementary strand).

In some cases (e.g., when Cas12J-1947455—also referred to herein as“ortholog #1”—as described herein is used), the PAM sequence of thenon-complementary strand is 5′-VTTR-3′ (where V is G, A, or C and R is Aor G)—see, e.g., FIG. 13A. Thus, in some cases, suitable PAMs caninclude GTTA, GTTG, ATTA, ATTG, CTTA, and CTTG.

In some cases (e.g., when Cas12J-2071242—also referred to herein as“ortholog #2”—as described herein is used), the PAM sequence of thenon-complementary strand is 5′-TBN-3′ (where B is T, C, or G)—see, e.g.,FIG. 13A. Thus, in some cases, suitable PAMs can include TTA, TTC, TTT,TTG, TCA, TCC, TCT, TCG, TGA, TGC, TGT, and TGG. In some embodiments(e.g., when Cas12J-2071242—also referred to herein as “ortholog #2”—asdescribed herein is used), the PAM sequence of the non-complementarystrand is 5′-TNN-3′.

In some cases (e.g., when Cas12J-3339380—also referred to herein as“ortholog #3”—as described herein is used), the PAM sequence of thenon-complementary strand is 5′-VTTB-3′ (where V is G, A, or C and whereB is T, C, or G)—see, e.g., FIG. 13A. Thus, in some cases, suitable PAMscan include GTTT, GTTC, GTTG, ATTT, ATTC, ATTG, CTTT, CTTC, CTTG.

In some cases (e.g., when Cas12J-3339380—also referred to herein as“ortholog #3”—as described herein is used), the PAM sequence of thenon-complementary strand is 5′-NTTN-3′. In some cases (e.g., whenCas12J-3339380—also referred to herein as “ortholog #3”—as describedherein is used), the PAM sequence of the non-complementary strand is5′-VTTN-3′ (where V is G, A, or C). In some embodiments (e.g., whenCas12J-3339380—also referred to herein as “ortholog #3”—as describedherein is used), the PAM sequence of the non-complementary strand is5′-VTTC-3′.

In some cases, different Cas12J proteins (i.e., Cas12J proteins fromvarious species) may be advantageous to use in the various providedmethods in order to capitalize on various enzymatic characteristics ofthe different Cas12J proteins (e.g., for different PAM sequencepreferences; for increased or decreased enzymatic activity; for anincreased or decreased level of cellular toxicity; to change the balancebetween NHEJ, homology-directed repair, single strand breaks, doublestrand breaks, etc.; to take advantage of a short total sequence; andthe like). Cas12J proteins from different species may require differentPAM sequences in the target DNA. Thus, for a particular Cas12J proteinof choice, the PAM sequence preference may be different than thesequences described above. Various methods (including in silico and/orwet lab methods) for identification of the appropriate PAM sequence areknown in the art and are routine, and any convenient method can be used.For example, PAM sequences described herein were identified using a PAMdepletion assay (e.g., see working examples below), but could also havebeen identified using a variety of different methods (includingcomputational analysis of sequencing data—as known in the art).

Cas12J Guide RNA

A nucleic acid that binds to a Cas12J protein, forming aribonucleoprotein complex (RNP), and targets the complex to a specificlocation within a target nucleic acid (e.g., a target DNA) is referredto herein as a “Cas12J guide RNA” or simply as a “guide RNA.” It is tobe understood that in some cases, a hybrid DNA/RNA can be made such thata Cas12J guide RNA includes DNA bases in addition to RNA bases, but theterm “Cas12J guide RNA” is still used to encompass such a moleculeherein.

A Cas12J guide RNA can be said to include two segments, a targetingsegment and a protein-binding segment. The protein-binding segment isalso referred to herein as the “constant region” of the guide RNA. Thetargeting segment of a Cas12J guide RNA includes a nucleotide sequence(a guide sequence) that is complementary to (and therefore hybridizeswith) a specific sequence (a target site) within a target nucleic acid(e.g., a target dsDNA, a target ssRNA, a target ssDNA, the complementarystrand of a double stranded target DNA, etc.). The protein-bindingsegment (or “protein-binding sequence”) interacts with (binds to) aCas12J polypeptide. The protein-binding segment of a subject Cas12Jguide RNA can include two complementary stretches of nucleotides thathybridize to one another to form a double stranded RNA duplex (dsRNAduplex). Site-specific binding and/or cleavage of a target nucleic acid(e.g., genomic DNA, ds DNA, RNA, etc.) can occur at locations (e.g.,target sequence of a target locus) determined by base-pairingcomplementarity between the Cas12J guide RNA (the guide sequence of theCas12J guide RNA) and the target nucleic acid.

A Cas12J guide RNA and a Cas12J protein (e.g., a wild-type Cas12Jprotein; a variant Cas12J protein; a fusion Cas12J polypeptide; etc.)form a complex (e.g., bind via non-covalent interactions). The Cas12Jguide RNA provides target specificity to the complex by including atargeting segment, which includes a guide sequence (a nucleotidesequence that is complementary to a sequence of a target nucleic acid).The Cas12J protein of the complex provides the site-specific activity(e.g., cleavage activity provided by the Cas12J protein and/or anactivity provided by the fusion partner in the case of a fusion Cas12Jprotein). In other words, the Cas12J protein is guided to a targetnucleic acid sequence (e.g. a target sequence) by virtue of itsassociation with the Cas12J guide RNA.

The “guide sequence” also referred to as the “targeting sequence” of aCas12J guide RNA can be modified so that the Cas12J guide RNA can targeta Cas12J protein (e.g., a naturally occurring Cas12J protein, a fusionCas12J polypeptide, and the like) to any desired sequence of any desiredtarget nucleic acid, with the exception (e.g., as described herein) thatthe PAM sequence can be taken into account. Thus, for example, a Cas12Jguide RNA can have a guide sequence with complementarity to (e.g., canhybridize to) a sequence in a nucleic acid in a eukaryotic cell, e.g., aviral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryoticchromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like.

Guide Sequence of a Cas12J Guide RNA

A subject Cas12J guide RNA includes a guide sequence (i.e., a targetingsequence), which is a nucleotide sequence that is complementary to asequence (a target site) in a target nucleic acid. In other words, theguide sequence of a Cas12J guide RNA can interact with a target nucleicacid (e.g., double stranded DNA (dsDNA), single stranded DNA (ssDNA),single stranded RNA (ssRNA), or double stranded RNA (dsRNA)) in asequence-specific manner via hybridization (i.e., base pairing). Theguide sequence of a Cas12J guide RNA can be modified (e.g., by geneticengineering)/designed to hybridize to any desired target sequence (e.g.,while taking the PAM into account, e.g., when targeting a dsDNA target)within a target nucleic acid (e.g., a eukaryotic target nucleic acidsuch as genomic DNA).

In some cases, the percent complementarity between the guide sequenceand the target site of the target nucleic acid is 60% or more (e.g., 65%or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% ormore, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). Insome cases, the percent complementarity between the guide sequence andthe target site of the target nucleic acid is 80% or more (e.g., 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100%). In some cases, the percent complementarity between the guidesequence and the target site of the target nucleic acid is 90% or more(e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%). Insome cases, the percent complementarity between the guide sequence andthe target site of the target nucleic acid is 100%.

In some cases, the percent complementarity between the guide sequenceand the target site of the target nucleic acid is 100% over the sevencontiguous 3′-most nucleotides of the target site of the target nucleicacid.

In some cases, the percent complementarity between the guide sequenceand the target site of the target nucleic acid is 60% or more (e.g., 70%or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100%) over 17 or more(e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more)contiguous nucleotides. In some cases, the percent complementaritybetween the guide sequence and the target site of the target nucleicacid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100%) over 17 or more (e.g., 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more) contiguousnucleotides. In some cases, the percent complementarity between theguide sequence and the target site of the target nucleic acid is 90% ormore (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%)over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more,22 or more) contiguous nucleotides. In some cases, the percentcomplementarity between the guide sequence and the target site of thetarget nucleic acid is 100% over 17 or more (e.g., 18 or more, 19 ormore, 20 or more, 21 or more, 22 or more) contiguous nucleotides.

In some cases, the percent complementarity between the guide sequenceand the target site of the target nucleic acid is 60% or more (e.g., 70%or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100%) over 19 or more(e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides. Insome cases, the percent complementarity between the guide sequence andthe target site of the target nucleic acid is 80% or more (e.g., 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more)contiguous nucleotides. In some cases, the percent complementaritybetween the guide sequence and the target site of the target nucleicacid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 ormore) contiguous nucleotides. In some cases, the percent complementaritybetween the guide sequence and the target site of the target nucleicacid is 100% over 19 or more (e.g., 20 or more, 21 or more, 22 or more)contiguous nucleotides.

In some cases, the percent complementarity between the guide sequenceand the target site of the target nucleic acid is 60% or more (e.g., 70%or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100%) over 17-25contiguous nucleotides. In some cases, the percent complementaritybetween the guide sequence and the target site of the target nucleicacid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100%) over 17-25 contiguousnucleotides. In some cases, the percent complementarity between theguide sequence and the target site of the target nucleic acid is 90% ormore (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%)over 17-25 contiguous nucleotides. In some cases, the percentcomplementarity between the guide sequence and the target site of thetarget nucleic acid is 100% over 17-25 contiguous nucleotides.

In some cases, the percent complementarity between the guide sequenceand the target site of the target nucleic acid is 60% or more (e.g., 70%or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100%) over 19-25contiguous nucleotides. In some cases, the percent complementaritybetween the guide sequence and the target site of the target nucleicacid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100%) over 19-25 contiguousnucleotides. In some cases, the percent complementarity between theguide sequence and the target site of the target nucleic acid is 90% ormore (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%)over 19-25 contiguous nucleotides. In some cases, the percentcomplementarity between the guide sequence and the target site of thetarget nucleic acid is 100% over 19-25 contiguous nucleotides.

In some cases, the guide sequence has a length in a range of from 17-30nucleotides (nt) (e.g., from 17-25, 17-22, 17-20, 19-30, 19-25, 19-22,19-20, 20-30, 20-25, or 20-22 nt). In some cases, the guide sequence hasa length in a range of from 17-25 nucleotides (nt) (e.g., from 17-22,17-20, 19-25, 19-22, 19-20, 20-25, or 20-22 nt). In some cases, theguide sequence has a length of 17 or more nt (e.g., 18 or more, 19 ormore, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has alength of 19 or more nt (e.g., 20 or more, 21 or more, or 22 or more nt;19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases,the guide sequence has a length of 17 nt. In some cases, the guidesequence has a length of 18 nt. In some cases, the guide sequence has alength of 19 nt. In some cases, the guide sequence has a length of 20nt. In some cases, the guide sequence has a length of 21 nt. In somecases, the guide sequence has a length of 22 nt. In some cases, theguide sequence has a length of 23 nt.

In some cases, the guide sequence (also referred to as a “spacersequence”) has a length of from 15 to 50 nucleotides (e.g., from 15nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt,from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from45 nt to 50 nt).

Protein-Binding Segment of a Cas12J Guide RNA

The protein-binding segment (the “constant region”) of a subject Cas12Jguide RNA interacts with a Cas12J protein. The Cas12J guide RNA guidesthe bound Cas12J protein to a specific nucleotide sequence within targetnucleic acid via the above-mentioned guide sequence. The protein-bindingsegment of a Cas12J guide RNA can include two stretches of nucleotidesthat are complementary to one another and hybridize to form a doublestranded RNA duplex (dsRNA duplex). Thus, in some cases, theprotein-binding segment includes a dsRNA duplex.

In some cases, the dsRNA duplex region includes a range of from 5-25base pairs (bp) (e.g., from 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 5-8,8-25, 8-22, 8-18, 8-15, 8-12, 12-25, 12-22, 12-18, 12-15, 13-25, 13-22,13-18, 13-15, 14-25, 14-22, 14-18, 14-15, 15-25, 15-22, 15-18, 17-25,17-22, or 17-18 bp, e.g., 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). Insome cases, the dsRNA duplex region includes a range of from 6-15 basepairs (bp) (e.g., from 6-12, 6-10, or 6-8 bp, e.g., 6 bp, 7 bp, 8 bp, 9bp, 10 bp, etc.). In some cases, the duplex region includes 5 or more bp(e.g., 6 or more, 7 or more, or 8 or more bp). In some cases, the duplexregion includes 6 or more bp (e.g., 7 or more, or 8 or more bp). In somecases, not all nucleotides of the duplex region are paired, andtherefore the duplex forming region can include a bulge. The term“bulge” herein is used to mean a stretch of nucleotides (which can beone nucleotide) that do not contribute to a double stranded duplex, butwhich are surround 5′ and 3′ by nucleotides that do contribute, and assuch a bulge is considered part of the duplex region. In some cases, thedsRNA includes 1 or more bulges (e.g., 2 or more, 3 or more, 4 or morebulges). In some cases, the dsRNA duplex includes 2 or more bulges(e.g., 3 or more, 4 or more bulges). In some cases, the dsRNA duplexincludes 1-5 bulges (e.g., 1-4, 1-3, 2-5, 2-4, or 2-3 bulges).

Thus, in some cases, the stretches of nucleotides that hybridize to oneanother to form the dsRNA duplex have 70%-100% complementarity (e.g.,75%-100%, 80%-10%, 85%-100%, 90%-100%, 95%-100% complementarity) withone another. In some cases, the stretches of nucleotides that hybridizeto one another to form the dsRNA duplex have 70%-100% complementarity(e.g., 75%-100%, 80%-10%, 85%-100%, 90%-100%, 95%-100% complementarity)with one another. In some cases, the stretches of nucleotides thathybridize to one another to form the dsRNA duplex have 85%-100%complementarity (e.g., 90%-100%, 95%-100% complementarity) with oneanother. In some cases, the stretches of nucleotides that hybridize toone another to form the dsRNA duplex have 70%-95% complementarity (e.g.,75%-95%, 80%-95%, 85%-95%, 90%-95% complementarity) with one another.

In other words, in some embodiments, the dsRNA duplex includes twostretches of nucleotides that have 70%-100% complementarity (e.g.,75%-100%, 80%-10%, 85%-100%, 90%-100%, 95%-100% complementarity) withone another. In some cases, the dsRNA duplex includes two stretches ofnucleotides that have 85%-100% complementarity (e.g., 90%-100%, 95%-100%complementarity) with one another. In some cases, the dsRNA duplexincludes two stretches of nucleotides that have 70%-95% complementarity(e.g., 75%-95%, 80%-95%, 85%-95%, 90%-95% complementarity) with oneanother.

The duplex region of a subject Cas12J guide RNA can include one or more(1, 2, 3, 4, 5, etc) mutations relative to a naturally occurring duplexregion. For example, in some cases a base pair can be maintained whilethe nucleotides contributing to the base pair from each segment can bedifferent. In some cases, the duplex region of a subject Cas12J guideRNA includes more paired bases, less paired bases, a smaller bulge, alarger bulge, fewer bulges, more bulges, or any convenient combinationthereof, as compared to a naturally occurring duplex region (of anaturally occurring Cas12J guide RNA).

Examples of various Cas9 guide RNAs can be found in the art, and in somecases variations similar to those introduced into Cas9 guide RNAs canalso be introduced into Cas12J guide RNAs of the present disclosure(e.g., mutations to the dsRNA duplex region, extension of the 5′ or 3′end for added stability for to provide for interaction with anotherprotein, and the like). For example, see Jinek et al., Science. 2012Aug. 17; 337(6096):816-21; Chylinski et al., RNA Biol. 2013 May;10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805; Hou et al.,Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Jinek et al.,Elife. 2013; 2:e00471; Pattanayak et al., Nat Biotechnol. 2013September; 31(9):839-43; Qi et al, Cell. 2013 Feb. 28; 152(5):1173-83;Wang et al., Cell. 2013 May 9; 153(4):910-8; Auer et al., Genome Res.2013 Oct. 31; Chen et al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e19;Cheng et al., Cell Res. 2013 October; 23(10):1163-71; Cho et al.,Genetics. 2013 November; 195(3):1177-80; DiCarlo et al., Nucleic AcidsRes. 2013 April; 41(7):4336-43; Dickinson et al., Nat Methods. 2013October; 10(10):1028-34; Ebina et al., Sci Rep. 2013; 3:2510; Fujii et.al, Nucleic Acids Res. 2013 Nov. 1; 41(20):e187; Hu et al., Cell Res.2013 November; 23(11):1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov.1; 41(20):e188; Larson et al., Nat Protoc. 2013 November; 8(11):2180-96;Mali et. at., Nat Methods. 2013 October; 10(10):957-63; Nakayama et al.,Genesis. 2013 December; 51(12):835-43; Ran et al., Nat Protoc. 2013November; 8(11):2281-308; Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9;Upadhyay et al., G3 (Bethesda). 2013 Dec. 9; 3(12):2233-8; Walsh et al.,Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15514-5; Xie et al., MolPlant. 2013 Oct. 9; Yang et al., Cell. 2013 Sep. 12; 154(6):1370-9;Briner et al., Mol Cell. 2014 Oct. 23; 56(2):333-9; and U.S. patents andpatent applications: U.S. Pat. Nos. 8,906,616; 8,895,308; 8,889,418;8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359;20140068797; 20140170753; 20140179006; 20140179770; 20140186843;20140186919; 20140186958; 20140189896; 20140227787; 20140234972;20140242664; 20140242699; 20140242700; 20140242702; 20140248702;20140256046; 20140273037; 20140273226; 20140273230; 20140273231;20140273232; 20140273233; 20140273234; 20140273235; 20140287938;20140295556; 20140295557; 20140298547; 20140304853; 20140309487;20140310828; 20140310830; 20140315985; 20140335063; 20140335620;20140342456; 20140342457; 20140342458; 20140349400; 20140349405;20140356867; 20140356956; 20140356958; 20140356959; 20140357523;20140357530; 20140364333; and 20140377868; all of which are herebyincorporated by reference in their entirety.

Examples of constant regions suitable for inclusion in a Cas12J guideRNA are provided in FIG. 7 (e.g., where T is substituted with U). ACas12J guide RNA can include a constant region having from 1 to 5nucleotide substitutions compared to any one of the nucleotide sequencesdepicted in FIG. 7. As one example, the constant region of a Cas12Jguide RNA can comprise the nucleotide sequence:GUCUCGACUAAUCGAGCAAUCGUUUGAGAUCUCUCC (SEQ ID NO: 83). As anotherexample, the constant region of a Cas12J guide RNA can comprise thenucleotide sequence: GUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGAC (SEQ ID NO:84). As another example, the constant region of a Cas12J guide RNA cancomprise the nucleotide sequence: GUCCCAGCGUACUGGGCAAUCAAUAGTCGUUUUGGU(SEQ ID NO: 85). As another example, the constant region of a Cas12Jguide RNA can comprise the nucleotide sequence:CACAGGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGAC (SEQ ID NO: 86). As anotherexample, the constant region of a Cas12J guide RNA can comprise thenucleotide sequence: UAAUGUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGAC (SEQ IDNO: 87). As another example, the constant region of a Cas12J guide RNAcan comprise the nucleotide sequence:AUUAACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGAC (SEQ ID NO: 88).

A Cas12J guide RNA constant region can include any one of the nucleotidesequences depicted in FIG. 8. A Cas12J guide RNA constant region caninclude a nucleotide sequence within the consensus sequence(s) depictedin FIG. 8.

The nucleotide sequences (with T substituted with U) can be combinedwith a spacer sequence (where the spacer sequence comprises a targetnucleic acid-binding sequence (“guide sequence”)) of choice that is from15 to 50 nucleotides (e.g., from 15 nucleotides (nt) to 20 nt, from 20nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40nt, from 40 nt to 45 nt, or from 45 nt to 50 nt in length). In somecases, the spacer sequence is 35-38 nucleotides in length. For example,any one of the nucleotide sequences (with T substituted with U) depictedin FIG. 7 can be included in a guide RNA comprising (N)n-constantregion, where N is any nucleotide and n is an integer from 15 to 50(e.g., from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from35 to 38, from 35 to 40, from 40 to 45, or from 45 to 50). The reversecomplement of any one of the nucleotide sequences depicted in FIG. 7(but with T substituted with U) can be included in a guide RNAcomprising constant region-(N)n, where N is any nucleotide and n is aninteger from 15 to 50 (e.g., from 15 to 20, from 20 to 25, from 25 to30, from 30 to 35, from 35 to 38, from 35 to 40, from 40 to 45, or from45 to 50).

As one example, a guide RNA can have the following nucleotide sequence:NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUCUCGACUAAUCGAGCAA UCGUUUGAGAUCUCUCC(SEQ ID NO: 89) or in some cases the reverse complement, where N is anynucleotide, e.g., where the stretch of Ns includes a target nucleicacid-binding sequence. As another example, a guide RNA can have thefollowing nucleotide sequence:NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUCGGAACGCUCAACGAUU GCCCCUCACGAGGGGAC(SEQ ID NO: 90) or in some cases the reverse complement, where N is anynucleotide, e.g., where the stretch of Ns includes a target nucleicacid-binding sequence.

As one example, a guide RNA can have the following nucleotide sequence:GUCUCGACUAAUCGAGCAAUCGUUUGAGAUCUCUCC (SEQ ID NO: 83)-‘guide sequence’(e.g., GUCUCGACUAAUCGAGCAAUCGUUUGAGAUCUCUCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 91), where the stretch of Ns representsthe guide sequence/targeting sequence and N is any nucleotide). Asanother example, a guide RNA can have the following nucleotide sequence:GGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGAC (SEQ ID NO: 177)-‘guide sequence’(e.g., GGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 92), where the stretch of Ns representsthe guide sequence/targeting sequence and N is any nucleotide).

As another example, a guide RNA can have the following nucleotidesequence: GUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGAC (SEQ ID NO: 84)-‘guidesequence’ (e.g., GUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 93), where the stretch of Ns representsthe guide sequence/targeting sequence and N is any nucleotide). Asanother example, a guide RNA can have the following nucleotide sequence:GUCCCCUCGUGAGGGGCAAUCGUUGAGCGUUCCGAC (SEQ ID NO: 169)-‘guide sequence’(e.g., GUCCCCUCGUGAGGGGCAAUCGUUGAGCGUUCCGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 94), where the stretch of Ns representsthe guide sequence/targeting sequence and N is any nucleotide).

As another example, a guide RNA can have the following nucleotidesequence: CACAGGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGAC (SEQ ID NO:86)-‘guide sequence’ (e.g.,CACAGGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 95), where the stretch of Nsrepresents the guide sequence/targeting sequence and N is anynucleotide). As another example, a guide RNA can have the followingnucleotide sequence: UAAUGUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGAC (SEQ IDNO: 87)-‘guide sequence’ (e.g.,UAAUGUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 96), where the stretch of Nsrepresents the guide sequence/targeting sequence and N is anynucleotide). As another example, a guide RNA can have the followingnucleotide sequence: AUUAACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGAC (SEQ IDNO: 88)-‘guide sequence’ (e.g.,AUUAACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 97), where the stretch of Nsrepresents the guide sequence/targeting sequence and N is anynucleotide).

Cas12J Guide Polynucleotides

In some cases, a nucleic acid that binds to a Cas12J protein, forming anucleic acid/Cas12J polypeptide complex, and that targets the complex toa specific location within a target nucleic acid (e.g., a target DNA)comprises ribonucleotides only, deoxyribonucleotides only, or a mixtureof ribonucleotides and deoxyribonucleotides. In some cases, a guidepolynucleotide comprises ribonucleotides only, and is referred to hereinas a “guide RNA.” In some cases, a guide polynucleotide comprisesdeoxyribonucleotides only, and is referred to herein as a “guide DNA.”In some cases, a guide polynucleotide comprises both ribonucleotides anddeoxyribonucleotides. A guide polynucleotide can comprise combinationsof ribonucleotide bases, deoxyribonucleotide bases, nucleotide analogs,modified nucleotides, and the like; and may further includenaturally-occurring backbone residues and/or linkages and/ornon-naturally-occurring backbone residues and/or linkages.

Cas12J Systems

The present disclosure provides a Cas12J system. A Cas12J system of thepresent disclosure can comprise: a) a Cas12J polypeptide of the presentdisclosure and a Cas12J guide RNA; b) a Cas12J polypeptide of thepresent disclosure, a Cas12J guide RNA, and a donor template nucleicacid; c) a Cas12J fusion polypeptide of the present disclosure and aCas12J guide RNA; d) a Cas12J fusion polypeptide of the presentdisclosure, a Cas12J guide RNA, and a donor template nucleic acid; e) anmRNA encoding a Cas12J polypeptide of the present disclosure; and aCas12J guide RNA; f) an mRNA encoding a Cas12J polypeptide of thepresent disclosure, a Cas12J guide RNA, and a donor template nucleicacid; g) an mRNA encoding a Cas12J fusion polypeptide of the presentdisclosure; and a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusionpolypeptide of the present disclosure, a Cas12J guide RNA, and a donortemplate nucleic acid; i) a recombinant expression vector comprising anucleotide sequence encoding a Cas12J polypeptide of the presentdisclosure and a nucleotide sequence encoding a Cas12J guide RNA; j) arecombinant expression vector comprising a nucleotide sequence encodinga Cas12J polypeptide of the present disclosure, a nucleotide sequenceencoding a Cas12J guide RNA, and a nucleotide sequence encoding a donortemplate nucleic acid; k) a recombinant expression vector comprising anucleotide sequence encoding a Cas12J fusion polypeptide of the presentdisclosure and a nucleotide sequence encoding a Cas12J guide RNA; 1) arecombinant expression vector comprising a nucleotide sequence encodinga Cas12J fusion polypeptide of the present disclosure, a nucleotidesequence encoding a Cas12J guide RNA, and a nucleotide sequence encodinga donor template nucleic acid; m) a first recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure, and a second recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J guide RNA; n) a firstrecombinant expression vector comprising a nucleotide sequence encodinga Cas12J polypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jguide RNA; and a donor template nucleic acid; o) a first recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jfusion polypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jguide RNA; p) a first recombinant expression vector comprising anucleotide sequence encoding a Cas12J fusion polypeptide of the presentdisclosure, and a second recombinant expression vector comprising anucleotide sequence encoding a Cas12J guide RNA; and a donor templatenucleic acid; q) a recombinant expression vector comprising a nucleotidesequence encoding a Cas12J polypeptide of the present disclosure, anucleotide sequence encoding a first Cas12J guide RNA, and a nucleotidesequence encoding a second Cas12J guide RNA; or r) a recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jfusion polypeptide of the present disclosure, a nucleotide sequenceencoding a first Cas12J guide RNA, and a nucleotide sequence encoding asecond Cas12J guide RNA; or some variation of one of (a) through (r).

Nucleic Acids

The present disclosure provides one or more nucleic acids comprising oneor more of: a donor polynucleotide sequence, a nucleotide sequenceencoding a Cas12J polypeptide (e.g., a wild type Cas12J protein, anickase Cas12J protein, a dCas12J protein, fusion Cas12J protein, andthe like), a Cas12J guide RNA, and a nucleotide sequence encoding aCas12J guide RNA. The present disclosure provides a nucleic acidcomprising a nucleotide sequence encoding a Cas12J fusion polypeptide.The present disclosure provides a recombinant expression vector thatcomprises a nucleotide sequence encoding a Cas12J polypeptide. Thepresent disclosure provides a recombinant expression vector thatcomprises a nucleotide sequence encoding a Cas12J fusion polypeptide.The present disclosure provides a recombinant expression vector thatcomprises: a) a nucleotide sequence encoding a Cas12J polypeptide; andb) a nucleotide sequence encoding a Cas12J guide RNA(s). The presentdisclosure provides a recombinant expression vector that comprises: a) anucleotide sequence encoding a Cas12J fusion polypeptide; and b) anucleotide sequence encoding a Cas12J guide RNA(s). In some cases, thenucleotide sequence encoding the Cas12J protein and/or the nucleotidesequence encoding the Cas12J guide RNA is operably linked to a promoterthat is operable in a cell type of choice (e.g., a prokaryotic cell, aeukaryotic cell, a plant cell, an animal cell, a mammalian cell, aprimate cell, a rodent cell, a human cell, etc.).

In some cases, a nucleotide sequence encoding a Cas12J polypeptide ofthe present disclosure is codon optimized. This type of optimization canentail a mutation of a Cas12J-encoding nucleotide sequence to mimic thecodon preferences of the intended host organism or cell while encodingthe same protein. Thus, the codons can be changed, but the encodedprotein remains unchanged. For example, if the intended target cell wasa human cell, a human codon-optimized Cas12J-encoding nucleotidesequence could be used. As another non-limiting example, if the intendedhost cell were a mouse cell, then a mouse codon-optimizedCas12J-encoding nucleotide sequence could be generated. As anothernon-limiting example, if the intended host cell were a plant cell, thena plant codon-optimized Cas12J-encoding nucleotide sequence could begenerated. As another non-limiting example, if the intended host cellwere an insect cell, then an insect codon-optimized Cas12J-encodingnucleotide sequence could be generated.

Codon usage tables are readily available, for example, at the “CodonUsage Database” available atwww[dot]kazusa[dot]or[dot]jp[forwardslash]codon. In some cases, anucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in a eukaryotic cell. In some cases, a nucleic acid of thepresent disclosure comprises a Cas12J polypeptide-encoding nucleotidesequence that is codon optimized for expression in an animal cell. Insome cases, a nucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in a fungus cell. In some cases, a nucleic acid of thepresent disclosure comprises a Cas12J polypeptide-encoding nucleotidesequence that is codon optimized for expression in a plant cell. In somecases, a nucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in a monocotyledonous plant species. In some cases, a nucleicacid of the present disclosure comprises a Cas12J polypeptide-encodingnucleotide sequence that is codon optimized for expression in adicotyledonous plant species. In some cases, a nucleic acid of thepresent disclosure comprises a Cas12J polypeptide-encoding nucleotidesequence that is codon optimized for expression in a gymnosperm plantspecies. In some cases, a nucleic acid of the present disclosurecomprises a Cas12J polypeptide-encoding nucleotide sequence that iscodon optimized for expression in an angiosperm plant species. In somecases, a nucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in a corn cell. In some cases, a nucleic acid of the presentdisclosure comprises a Cas12J polypeptide-encoding nucleotide sequencethat is codon optimized for expression in a soybean cell. In some cases,a nucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in a rice cell. In some cases, a nucleic acid of the presentdisclosure comprises a Cas12J polypeptide-encoding nucleotide sequencethat is codon optimized for expression in a wheat cell. In some cases, anucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in a cotton cell. In some cases, a nucleic acid of thepresent disclosure comprises a Cas12J polypeptide-encoding nucleotidesequence that is codon optimized for expression in a sorghum cell. Insome cases, a nucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in an alfalfa cell. In some cases, a nucleic acid of thepresent disclosure comprises a Cas12J polypeptide-encoding nucleotidesequence that is codon optimized for expression in a sugar cane cell. Insome cases, a nucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in an Arabidopsis cell. In some cases, a nucleic acid of thepresent disclosure comprises a Cas12J polypeptide-encoding nucleotidesequence that is codon optimized for expression in a tomato cell. Insome cases, a nucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in a cucumber cell. In some cases, a nucleic acid of thepresent disclosure comprises a Cas12J polypeptide-encoding nucleotidesequence that is codon optimized for expression in a potato cell. Insome cases, a nucleic acid of the present disclosure comprises a Cas12Jpolypeptide-encoding nucleotide sequence that is codon optimized forexpression in an algae cell.

The present disclosure provides one or more recombinant expressionvectors that include (in different recombinant expression vectors insome cases, and in the same recombinant expression vector in somecases): (i) a nucleotide sequence of a donor template nucleic acid(where the donor template comprises a nucleotide sequence havinghomology to a target sequence of a target nucleic acid (e.g., a targetgenome)); (ii) a nucleotide sequence that encodes a Cas12J guide RNAthat hybridizes to a target sequence of the target locus of the targetedgenome (e.g., operably linked to a promoter that is operable in a targetcell such as a eukaryotic cell); and (iii) a nucleotide sequenceencoding a Cas12J protein (e.g., operably linked to a promoter that isoperable in a target cell such as a eukaryotic cell). The presentdisclosure provides one or more recombinant expression vectors thatinclude (in different recombinant expression vectors in some cases, andin the same recombinant expression vector in some cases): (i) anucleotide sequence of a donor template nucleic acid (where the donortemplate comprises a nucleotide sequence having homology to a targetsequence of a target nucleic acid (e.g., a target genome)); and (ii) anucleotide sequence that encodes a Cas12J guide RNA that hybridizes to atarget sequence of the target locus of the targeted genome (e.g.,operably linked to a promoter that is operable in a target cell such asa eukaryotic cell). The present disclosure provides one or morerecombinant expression vectors that include (in different recombinantexpression vectors in some cases, and in the same recombinant expressionvector in some cases): (i) a nucleotide sequence that encodes a Cas12Jguide RNA that hybridizes to a target sequence of the target locus ofthe targeted genome (e.g., operably linked to a promoter that isoperable in a target cell such as a eukaryotic cell); and (ii) anucleotide sequence encoding a Cas12J protein (e.g., operably linked toa promoter that is operable in a target cell such as a eukaryotic cell).

Suitable expression vectors include viral expression vectors (e.g. viralvectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Liet al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., GeneTher 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamotoet al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associatedvirus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998,Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., InvestOpthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al.,Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski etal., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like. In some cases, a recombinant expressionvector of the present disclosure is a recombinant adeno-associated virus(AAV) vector. In some cases, a recombinant expression vector of thepresent disclosure is a recombinant lentivirus vector. In some cases, arecombinant expression vector of the present disclosure is a recombinantretroviral vector.

For plant applications, viral vectors based on Tobamoviruses,Potexviruses, Potyviruses, Tobraviruses, Tombusviruses, Geminiviruses,Bromoviruses, Carmoviruses, Alfamoviruses, or Cucumoviruses can be used.See, e.g., Peyret and Lomonossoff (2015) Plant Biotechnol. J. 13:1121.Suitable Tobamovirus vectors include, for example, a tomato mosaic virus(ToMV) vector, a tobacco mosaic virus (TMV) vector, a tobacco mild greenmosaic virus (TMGMV) vector, a pepper mild mottle virus (PMMoV) vector,a paprika mild mottle virus (PaMMV) vector, a cucumber green mottlemosaic virus (CGMMV) vector, a kyuri green mottle mosaic virus (KGMMV)vector, a hibiscus latent fort pierce virus (HLFPV) vector, anodontoglossum ringspot virus (ORSV) vector, a rehmannia mosaic virus(ReMV) vector, a Sammon's opuntia virus (SOV) vector, a wasabi mottlevirus (WMoV) vector, a youcai mosaic virus (YoMV) vector, a sunn-hempmosaic virus (SHMV) vector, and the like. Suitable Potexvirus vectorsinclude, for example, a potato virus X (PVX) vector, a potatoaucubamosaicvirus (PAMV) vector, an Alstroemeria virus X (AlsVX) vector,a cactus virus X (CVX) vector, a Cymbidium mosaic virus (CymMV) vector,a hosta virus X (HVX) vector, a lily virus X (LVX) vector, a Narcissusmosaic virus (NMV) vector, a Nerine virus X (NVX) vector, a Plantagoasiatica mosaic virus (PlAMV) vector, a strawberry mild yellow edgevirus (SMYEV) vector, a tulip virus X (TVX) vector, a white clovermosaic virus (WClMV) vector, a bamboo mosaic virus (BaMV) vector, andthe like. Suitable Potyvirus vectors include, for example, a potatovirus Y (PVY) vector, a bean common mosaic virus (BCMV) vector, a cloveryellow vein virus (ClYVV) vector, an East Asian Passiflora virus (EAPV)vector, a Freesia mosaic virus (FreMV) vector, a Japanese yam mosaicvirus (JYMV) vector, a lettuce mosaic virus (LMV) vector, a Maize dwarfmosaic virus (MDMV) vector, an onion yellow dwarf virus (OYDV) vector, apapaya ringspot virus (PRSV) vector, a pepper mottle virus (PepMoV)vector, a Perilla mottle virus (PerMoV) vector, a plum pox virus (PPV)vector, a potato virus A (PVA) vector, a sorghum mosaic virus (SrMV)vector, a soybean mosaic virus (SMV) vector, a sugarcane mosaic virus(SCMV) vector, a tulip mosaic virus (TulMV) vector, a turnip mosaicvirus (TuMV) vector, a watermelon mosaic virus (WMV) vector, a zucchiniyellow mosaic virus (ZYMV) vector, a tobacco etch virus (TEV) vector,and the like. Suitable Tobravirus vectors include, for example, atobacco rattle virus (TRV) vector and the like. Suitable Tombusvirusvectors include, for example, a tomato bushy stunt virus (TBSV) vector,an eggplant mottled crinkle virus (EMCV) vector, a grapevine Algerianlatent virus (GALV) vector, and the like. Suitable Cucumovirus vectorsinclude, for example, a cucumber mosaic virus (CMV) vector, a peanutstunt virus (PSV) vector, a tomato aspermy virus (TAV) vector, and thelike. Suitable Bromovirus vectors include, for example, a brome mosaicvirus (BMV) vector, a cowpea chlorotic mottle virus (CCMV) vector, andthe like. Suitable Carmovirus vectors include, for example, a carnationmottle virus (CarMV) vector, a melon necrotic spot virus (MNSV) vector,a pea stem necrotic virus (PSNV) vector, a turnip crinkle virus (TCV)vector, and the like. Suitable Alfamovirus vectors include, for example,an alfalfa mosaic virus (AMV) vector, and the like.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector.

In some embodiments, a nucleotide sequence encoding a Cas12J guide RNAis operably linked to a control element, e.g., a transcriptional controlelement, such as a promoter. In some embodiments, a nucleotide sequenceencoding a Cas12J protein or a Cas12J fusion polypeptide is operablylinked to a control element, e.g., a transcriptional control element,such as a promoter.

The transcriptional control element can be a promoter. In some cases,the promoter is a constitutively active promoter. In some cases, thepromoter is a regulatable promoter. In some cases, the promoter is aninducible promoter. In some cases, the promoter is a tissue-specificpromoter. In some cases, the promoter is a cell type-specific promoter.In some cases, the transcriptional control element (e.g., the promoter)is functional in a targeted cell type or targeted cell population. Forexample, in some cases, the transcriptional control element can befunctional in eukaryotic cells, e.g., hematopoietic stem cells (e.g.,mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+)cell, etc.).

Non-limiting examples of eukaryotic promoters (promoters functional in aeukaryotic cell) include EF1α, those from cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, early andlate SV40, long terminal repeats (LTRs) from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art. The expressionvector may also contain a ribosome binding site for translationinitiation and a transcription terminator. The expression vector mayalso include appropriate sequences for amplifying expression. Theexpression vector may also include nucleotide sequences encoding proteintags (e.g., 6×His tag, hemagglutinin tag, fluorescent protein, etc.)that can be fused to the Cas12J protein, thus resulting in a fusionCas12J polypeptide.

In some embodiments, a nucleotide sequence encoding a Cas12J guide RNAand/or a Cas12J fusion polypeptide is operably linked to an induciblepromoter. In some embodiments, a nucleotide sequence encoding a Cas12Jguide RNA and/or a Cas12J fusion protein is operably linked to aconstitutive promoter.

A promoter can be a constitutively active promoter (i.e., a promoterthat is constitutively in an active/“ON” state), it may be an induciblepromoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”,is controlled by an external stimulus, e.g., the presence of aparticular temperature, compound, or protein.), it may be a spatiallyrestricted promoter (i.e., transcriptional control element, enhancer,etc.)(e.g., tissue specific promoter, cell type specific promoter,etc.), and it may be a temporally restricted promoter (i.e., thepromoter is in the “ON” state or “OFF” state during specific stages ofembryonic development or during specific stages of a biological process,e.g., hair follicle cycle in mice).

Suitable promoters can be derived from viruses and can therefore bereferred to as viral promoters, or they can be derived from anyorganism, including prokaryotic or eukaryotic organisms. Suitablepromoters can be used to drive expression by any RNA polymerase (e.g.,pol I, pol II, pol III). Exemplary promoters include, but are notlimited to the SV40 early promoter, mouse mammary tumor virus longterminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP);a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishiet al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), ahuman H1 promoter (H1), and the like.

In some cases, a nucleotide sequence encoding a Cas12J guide RNA isoperably linked to (under the control of) a promoter operable in aeukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an H1promoter, and the like). As would be understood by one of ordinary skillin the art, when expressing an RNA (e.g., a guide RNA) from a nucleicacid (e.g., an expression vector) using a U6 promoter (e.g., in aeukaryotic cell), or another PolIII promoter, the RNA may need to bemutated if there are several Ts in a row (coding for Us in the RNA).This is because a string of Ts (e.g., 5 Ts) in DNA can act as aterminator for polymerase III (PolIII). Thus, in order to ensuretranscription of a guide RNA in a eukaryotic cell it may sometimes benecessary to modify the sequence encoding the guide RNA to eliminateruns of Ts. In some cases, a nucleotide sequence encoding a Cas12Jprotein (e.g., a wild type Cas12J protein, a nickase Cas12J protein, adCas12J protein, a fusion Cas12J protein and the like) is operablylinked to a promoter operable in a eukaryotic cell (e.g., a CMVpromoter, an EF1α promoter, an estrogen receptor-regulated promoter, andthe like).

Examples of inducible promoters include, but are not limited to T7 RNApolymerase promoter, T3 RNA polymerase promoter,Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter,lactose induced promoter, heat shock promoter, Tetracycline-regulatedpromoter, Steroid-regulated promoter, Metal-regulated promoter, estrogenreceptor-regulated promoter, etc. Inducible promoters can therefore beregulated by molecules including, but not limited to, doxycycline;estrogen and/or an estrogen analog; IPTG; etc.

Inducible promoters suitable for use include any inducible promoterdescribed herein or known to one of ordinary skill in the art. Examplesof inducible promoters include, without limitation,chemically/biochemically-regulated and physically-regulated promoterssuch as alcohol-regulated promoters, tetracycline-regulated promoters(e.g., anhydrotetracycline (aTc)-responsive promoters and othertetracycline-responsive promoter systems, which include a tetracyclinerepressor protein (tetR), a tetracycline operator sequence (tetO) and atetracycline transactivator fusion protein (tTA)), steroid-regulatedpromoters (e.g., promoters based on the rat glucocorticoid receptor,human estrogen receptor, moth ecdysone receptors, and promoters from thesteroid/retinoid/thyroid receptor superfamily), metal-regulatedpromoters (e.g., promoters derived from metallothionein (proteins thatbind and sequester metal ions) genes from yeast, mouse and human),pathogenesis-regulated promoters (e.g., induced by salicylic acid,ethylene or benzothiadiazole (BTH)), temperature/heat-induciblepromoters (e.g., heat shock promoters), and light-regulated promoters(e.g., light responsive promoters from plant cells).

In some cases, the promoter is a spatially restricted promoter (i.e.,cell type specific promoter, tissue specific promoter, etc.) such thatin a multi-cellular organism, the promoter is active (i.e., “ON”) in asubset of specific cells. Spatially restricted promoters may also bereferred to as enhancers, transcriptional control elements, controlsequences, etc. Any convenient spatially restricted promoter may be usedas long as the promoter is functional in the targeted host cell (e.g.,eukaryotic cell; prokaryotic cell).

In some cases, the promoter is a reversible promoter. Suitablereversible promoters, including reversible inducible promoters are knownin the art. Such reversible promoters may be isolated and derived frommany organisms, e.g., eukaryotes and prokaryotes. Modification ofreversible promoters derived from a first organism for use in a secondorganism, e.g., a first prokaryote and a second a eukaryote, a firsteukaryote and a second a prokaryote, etc., is well known in the art.Such reversible promoters, and systems based on such reversiblepromoters but also comprising additional control proteins, include, butare not limited to, alcohol regulated promoters (e.g., alcoholdehydrogenase I (alcA) gene promoter, promoters responsive to alcoholtransactivator proteins (AlcR), etc.), tetracycline regulated promoters,(e.g., promoter systems including TetActivators, TetON, TetOFF, etc.),steroid regulated promoters (e.g., rat glucocorticoid receptor promotersystems, human estrogen receptor promoter systems, retinoid promotersystems, thyroid promoter systems, ecdysone promoter systems,mifepristone promoter systems, etc.), metal regulated promoters (e.g.,metallothionein promoter systems, etc.), pathogenesis-related regulatedpromoters (e.g., salicylic acid regulated promoters, ethylene regulatedpromoters, benzothiadiazole regulated promoters, etc.), temperatureregulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70,HSP-90, soybean heat shock promoter, etc.), light regulated promoters,synthetic inducible promoters, and the like.

RNA polymerase III (Pol III) promoters can be used to drive theexpression of non-protein coding RNA molecules (e.g., guide RNAs). Insome cases, a suitable promoter is a Pol III promoter. In some cases, aPol III promoter is operably linked to a nucleotide sequence encoding aguide RNA (gRNA). In some cases, a Pol III promoter is operably linkedto a nucleotide sequence encoding a single-guide RNA (sgRNA). In somecases, a Pol III promoter is operably linked to a nucleotide sequenceencoding a CRISPR RNA (crRNA). In some cases, a Pol III promoter isoperably linked to a nucleotide sequence encoding a encoding a tracrRNA.

Non-limiting examples of Pol III promoters include a U6 promoter, an H1promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI promoter, a tRNApromoter, and a 7SK promoter. See, for example, Schramm and Hernandez(2002) Genes & Development 16:2593-2620. In some cases, a Pol IIIpromoter is selected from the group consisting of a U6 promoter, an H1promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI promoter, a tRNApromoter, and a 7SK promoter. In some cases, a guide RNA-encodingnucleotide sequence is operably linked to a promoter selected from thegroup consisting of a U6 promoter, an H1 promoter, a 5S promoter, anAdenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. Insome cases, a single-guide RNA-encoding nucleotide sequence is operablylinked to a promoter selected from the group consisting of a U6promoter, an H1 promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAIpromoter, a tRNA promoter, and a 7SK promoter.

Examples describing a promoter that can be used herein in connectionwith expression in plants, plant tissues, and plant cells include, butare not limited to, promoters described in: U.S. Pat. No. 6,437,217(maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter),U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362(maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S.Pat. No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos.5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat.No. 6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (riceactin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No.5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (lightinducible promoters), U.S. Pat. No. 6,140,078 (salt induciblepromoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), U.S.Pat. No. 6,175,060 (phosphorus deficiency inducible promoters), U.S.Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. patent applicationSer. No. 09/757,089 (maize chloroplast aldolase promoter). Additionalpromoters that can find use include a nopaline synthase (NOS) promoter(Ebert et al., 1987), the octopine synthase (OCS) promoter (which iscarried on tumor-inducing plasmids of Agrobacterium tumefaciens), thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Spromoter (Lawton et al. Plant Molecular Biology (1987) 9: 315-324), theCaMV 35S promoter (Odell et al., Nature (1985) 313: 810-812), thefigwort mosaic virus 35S-promoter (U.S. Pat. Nos. 6,051,753; 5,378,619),the sucrose synthase promoter (Yang and Russell, Proceedings of theNational Academy of Sciences, USA (1990) 87: 4144-4148), the R genecomplex promoter (Chandler et al., Plant Cell (1989) 1: 1175-1183), andthe chlorophyll a/b binding protein gene promoter, PClSV (U.S. Pat. No.5,850,019), and AGRtu.nos (GenBank Accession V00087; Depicker et al.,Journal of Molecular and Applied Genetics (1982) 1:561-573; Bevan etal., 1983) promoters.

Methods of introducing a nucleic acid (e.g., a nucleic acid comprising adonor polynucleotide sequence, one or more nucleic acids encoding aCas12J protein and/or a Cas12J guide RNA, and the like) into a host cellare known in the art, and any convenient method can be used to introducea nucleic acid (e.g., an expression construct) into a cell. Suitablemethods include e.g., viral infection, transfection, lipofection,electroporation, calcium phosphate precipitation, polyethyleneimine(PEI)-mediated transfection, DEAE-dextran mediated transfection,liposome-mediated transfection, particle gun technology, calciumphosphate precipitation, direct microinjection, nanoparticle-mediatednucleic acid delivery, and the like.

Introducing the recombinant expression vector into cells can occur inany culture media and under any culture conditions that promote thesurvival of the cells. Introducing the recombinant expression vectorinto a target cell can be carried out in vivo or ex vivo. Introducingthe recombinant expression vector into a target cell can be carried outin vitro.

In some embodiments, a Cas12J protein can be provided as RNA. The RNAcan be provided by direct chemical synthesis or may be transcribed invitro from a DNA (e.g., encoding the Cas12J protein). Once synthesized,the RNA may be introduced into a cell by any of the well-knowntechniques for introducing nucleic acids into cells (e.g.,microinjection, electroporation, transfection, etc.).

Nucleic acids may be provided to the cells using well-developedtransfection techniques; see, e.g. Angel and Yanik (2010) PLoS ONE 5(7):e11756, and the commercially available TransMessenger® reagents fromQiagen, Stemfect™ RNA Transfection Kit from Stemgent, and TransIT®-mRNATransfection Kit from Minis Bio LLC. See also Beumer et al. (2008) PNAS105(50):19821-19826.

Vectors may be provided directly to a target host cell. In other words,the cells are contacted with vectors comprising the subject nucleicacids (e.g., recombinant expression vectors having the donor templatesequence and encoding the Cas12J guide RNA; recombinant expressionvectors encoding the Cas12J protein; etc.) such that the vectors aretaken up by the cells. Methods for contacting cells with nucleic acidvectors that are plasmids, include electroporation, calcium chloridetransfection, microinjection, and lipofection are well known in the art.For viral vector delivery, cells can be contacted with viral particlescomprising the subject viral expression vectors.

Retroviruses, for example, lentiviruses, are suitable for use in methodsof the present disclosure. Commonly used retroviral vectors are“defective”, i.e. unable to produce viral proteins required forproductive infection. Rather, replication of the vector requires growthin a packaging cell line. To generate viral particles comprising nucleicacids of interest, the retroviral nucleic acids comprising the nucleicacid are packaged into viral capsids by a packaging cell line. Differentpackaging cell lines provide a different envelope protein (ecotropic,amphotropic or xenotropic) to be incorporated into the capsid, thisenvelope protein determining the specificity of the viral particle forthe cells (ecotropic for murine and rat; amphotropic for most mammaliancell types including human, dog and mouse; and xenotropic for mostmammalian cell types except murine cells). The appropriate packagingcell line may be used to ensure that the cells are targeted by thepackaged viral particles. Methods of introducing subject vectorexpression vectors into packaging cell lines and of collecting the viralparticles that are generated by the packaging lines are well known inthe art. Nucleic acids can also introduced by direct micro-injection(e.g., injection of RNA).

Vectors used for providing the nucleic acids encoding Cas12J guide RNAand/or a Cas12J polypeptide to a target host cell can include suitablepromoters for driving the expression, that is, transcriptionalactivation, of the nucleic acid of interest. In other words, in somecases, the nucleic acid of interest will be operably linked to apromoter. This may include ubiquitously acting promoters, for example,the CMV-β-actin promoter, or inducible promoters, such as promoters thatare active in particular cell populations or that respond to thepresence of drugs such as tetracycline. By transcriptional activation,it is intended that transcription will be increased above basal levelsin the target cell by 10 fold, by 100 fold, more usually by 1000 fold.In addition, vectors used for providing a nucleic acid encoding a Cas12Jguide RNA and/or a Cas12J protein to a cell may include nucleic acidsequences that encode for selectable markers in the target cells, so asto identify cells that have taken up the Cas12J guide RNA and/or Cas12Jprotein.

A nucleic acid comprising a nucleotide sequence encoding a Cas12Jpolypeptide, or a Cas12J fusion polypeptide, is in some cases an RNA.Thus, a Cas12J fusion protein can be introduced into cells as RNA.Methods of introducing RNA into cells are known in the art and mayinclude, for example, direct injection, transfection, or any othermethod used for the introduction of DNA. A Cas12J protein may instead beprovided to cells as a polypeptide. Such a polypeptide may optionally befused to a polypeptide domain that increases solubility of the product.The domain may be linked to the polypeptide through a defined proteasecleavage site, e.g. a TEV sequence, which is cleaved by TEV protease.The linker may also include one or more flexible sequences, e.g. from 1to 10 glycine residues. In some embodiments, the cleavage of the fusionprotein is performed in a buffer that maintains solubility of theproduct, e.g. in the presence of from 0.5 to 2 M urea, in the presenceof polypeptides and/or polynucleotides that increase solubility, and thelike. Domains of interest include endosomolytic domains, e.g. influenzaHA domain; and other polypeptides that aid in production, e.g. IF2domain, GST domain, GRPE domain, and the like. The polypeptide may beformulated for improved stability. For example, the peptides may bePEGylated, where the polyethyleneoxy group provides for enhancedlifetime in the blood stream.

Additionally or alternatively, a Cas12J polypeptide of the presentdisclosure may be fused to a polypeptide permeant domain to promoteuptake by the cell. A number of permeant domains are known in the artand may be used in the non-integrating polypeptides of the presentdisclosure, including peptides, peptidomimetics, and non-peptidecarriers. For example, a permeant peptide may be derived from the thirdalpha helix of Drosophila melanogaster transcription factorAntennapaedia, referred to as penetratin, which comprises the amino acidsequence RQIKIWFQNRRMKWKK (SEQ ID NO: 68). As another example, thepermeant peptide comprises the HIV-1 tat basic region amino acidsequence, which may include, for example, amino acids 49-57 ofnaturally-occurring tat protein. Other permeant domains includepoly-arginine motifs, for example, the region of amino acids 34-56 ofHIV-1 rev protein, nona-arginine, octa-arginine, and the like. (See, forexample, Futaki et al. (2003) Curr Protein Pept Sci. 2003 April; 4(2):87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A 2000Nov. 21; 97(24):13003-8; published U.S. Patent applications 20030220334;20030083256; 20030032593; and 20030022831, herein specificallyincorporated by reference for the teachings of translocation peptidesand peptoids). The nona-arginine (R9) sequence is one of the moreefficient PTDs that have been characterized (Wender et al. 2000; Uemuraet al. 2002). The site at which the fusion is made may be selected inorder to optimize the biological activity, secretion or bindingcharacteristics of the polypeptide. The optimal site will be determinedby routine experimentation.

As noted above, in some cases, the target cell is a plant cell. Numerousmethods for transforming chromosomes or plastids in a plant cell with arecombinant nucleic acid are known in the art, which can be usedaccording to methods of the present application to produce a transgenicplant cell and/or a transgenic plant. Any suitable method or techniquefor transformation of a plant cell known in the art can be used.Effective methods for transformation of plants include bacteriallymediated transformation, such as Agrobacterium-mediated orRhizobium-mediated transformation and microprojectilebombardment-mediated transformation. A variety of methods are known inthe art for transforming explants with a transformation vector viabacterially mediated transformation or microprojectile bombardment andthen subsequently culturing, etc., those explants to regenerate ordevelop transgenic plants. Other methods for plant transformation, suchas microinjection, electroporation, vacuum infiltration, pressure,sonication, silicon carbide fiber agitation, PEG-mediatedtransformation, etc., are also known in the art. Transgenic plantsproduced by these transformation methods can be chimeric or non-chimericfor the transformation event depending on the methods and explants used.

Methods of transforming plant cells are well known by persons ofordinary skill in the art. For instance, specific instructions fortransforming plant cells by microprojectile bombardment with particlescoated with recombinant DNA (e.g., biolistic transformation) are foundin U.S. Pat. Nos. 5,550,318; 5,538,880 6,160,208; 6,399,861; and6,153,812 and Agrobacterium-mediated transformation is described in U.S.Pat. Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871;5,463,174; and 5,188,958. Additional methods for transforming plants canbe found in, for example, Compendium of Transgenic Crop Plants (2009)Blackwell Publishing. Any appropriate method known to those skilled inthe art can be used to transform a plant cell with any of the nucleicacids provided herein.

A Cas12J polypeptide of the present disclosure may be produced in vitroor by eukaryotic cells or by prokaryotic cells, and it may be furtherprocessed by unfolding, e.g. heat denaturation, dithiothreitolreduction, etc. and may be further refolded, using methods known in theart.

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acylation, acetylation,carboxylation, amidation, etc. Also included are modifications ofglycosylation, e.g. those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g. by exposing the polypeptide to enzymes whichaffect glycosylation, such as mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences that have phosphorylated amino acidresidues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

Also suitable for inclusion in embodiments of the present disclosure arenucleic acids (e.g., encoding a Cas12J guide RNA, encoding a Cas12Jfusion protein, etc.) and proteins (e.g., a Cas12J fusion proteinderived from a wild type protein or a variant protein) that have beenmodified using ordinary molecular biological techniques and syntheticchemistry so as to improve their resistance to proteolytic degradation,to change the target sequence specificity, to optimize solubilityproperties, to alter protein activity (e.g., transcription modulatoryactivity, enzymatic activity, etc.) or to render them more suitable.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g. D-amino acids ornon-naturally occurring synthetic amino acids. D-amino acids may besubstituted for some or all of the amino acid residues.

A Cas12J polypeptide of the present disclosure may be prepared by invitro synthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus, e.g., cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

A Cas12J polypeptide of the present disclosure may also be isolated andpurified in accordance with conventional methods of recombinantsynthesis. A lysate may be prepared of the expression host and thelysate purified using high performance liquid chromatography (HPLC),exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. For the most part, the compositionswhich are used will comprise 20% or more by weight of the desiredproduct, more usually 75% or more by weight, preferably 95% or more byweight, and for therapeutic purposes, usually 99.5% or more by weight,in relation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein. Thus, in some cases, a Cas12J polypeptide, or aCas12J fusion polypeptide, of the present disclosure is at least 80%pure, at least 85% pure, at least 90% pure, at least 95% pure, at least98% pure, or at least 99% pure (e.g., free of contaminants, non-Cas12Jproteins or other macromolecules, etc.).

To induce cleavage or any desired modification to a target nucleic acid(e.g., genomic DNA), or any desired modification to a polypeptideassociated with target nucleic acid, the Cas12J guide RNA and/or theCas12J polypeptide of the present disclosure and/or the donor templatesequence, whether they be introduced as nucleic acids or polypeptides,are provided to the cells for about 30 minutes to about 24 hours, e.g.,1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20hours, or any other period from about 30 minutes to about 24 hours,which may be repeated with a frequency of about every day to about every4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any otherfrequency from about every day to about every four days. The agent(s)may be provided to the subject cells one or more times, e.g. one time,twice, three times, or more than three times, and the cells allowed toincubate with the agent(s) for some amount of time following eachcontacting event e.g. 16-24 hours, after which time the media isreplaced with fresh media and the cells are cultured further.

In cases in which two or more different targeting complexes are providedto the cell (e.g., two different Cas12J guide RNAs that arecomplementary to different sequences within the same or different targetnucleic acid), the complexes may be provided simultaneously (e.g. as twopolypeptides and/or nucleic acids), or delivered simultaneously.Alternatively, they may be provided consecutively, e.g. the targetingcomplex being provided first, followed by the second targeting complex,etc. or vice versa.

To improve the delivery of a DNA vector into a target cell, the DNA canbe protected from damage and its entry into the cell facilitated, forexample, by using lipoplexes and polyplexes. Thus, in some cases, anucleic acid of the present disclosure (e.g., a recombinant expressionvector of the present disclosure) can be covered with lipids in anorganized structure like a micelle or a liposome. When the organizedstructure is complexed with DNA it is called a lipoplex. There are threetypes of lipids, anionic (negatively-charged), neutral, or cationic(positively-charged). Lipoplexes that utilize cationic lipids haveproven utility for gene transfer. Cationic lipids, due to their positivecharge, naturally complex with the negatively charged DNA. Also, as aresult of their charge, they interact with the cell membrane.Endocytosis of the lipoplex then occurs, and the DNA is released intothe cytoplasm. The cationic lipids also protect against degradation ofthe DNA by the cell.

Complexes of polymers with DNA are called polyplexes. Most polyplexesconsist of cationic polymers and their production is regulated by ionicinteractions. One large difference between the methods of action ofpolyplexes and lipoplexes is that polyplexes cannot release their DNAload into the cytoplasm, so to this end, co-transfection withendosome-lytic agents (to lyse the endosome that is made duringendocytosis) such as inactivated adenovirus must occur. However, this isnot always the case; polymers such as polyethylenimine have their ownmethod of endosome disruption as does chitosan and trimethylchitosan.

Dendrimers, a highly branched macromolecule with a spherical shape, maybe also be used to genetically modify stem cells. The surface of thedendrimer particle may be functionalized to alter its properties. Inparticular, it is possible to construct a cationic dendrimer (i.e., onewith a positive surface charge). When in the presence of geneticmaterial such as a DNA plasmid, charge complementarity leads to atemporary association of the nucleic acid with the cationic dendrimer.On reaching its destination, the dendrimer-nucleic acid complex can betaken up into a cell by endocytosis.

In some cases, a nucleic acid of the disclosure (e.g., an expressionvector) includes an insertion site for a guide sequence of interest. Forexample, a nucleic acid can include an insertion site for a guidesequence of interest, where the insertion site is immediately adjacentto a nucleotide sequence encoding the portion of a Cas12J guide RNA thatdoes not change when the guide sequence is changed to hybridized to adesired target sequence (e.g., sequences that contribute to the Cas12Jbinding aspect of the guide RNA, e.g., the sequences that contribute tothe dsRNA duplex(es) of the Cas12J guide RNA—this portion of the guideRNA can also be referred to as the ‘scaffold’ or ‘constant region’ ofthe guide RNA). Thus, in some cases, a subject nucleic acid (e.g., anexpression vector) includes a nucleotide sequence encoding a Cas12Jguide RNA, except that the portion encoding the guide sequence portionof the guide RNA is an insertion sequence (an insertion site). Aninsertion site is any nucleotide sequence used for the insertion of thedesired sequence. “Insertion sites” for use with various technologiesare known to those of ordinary skill in the art and any convenientinsertion site can be used. An insertion site can be for any method formanipulating nucleic acid sequences. For example, in some cases theinsertion site is a multiple cloning site (MCS) (e.g., a site includingone or more restriction enzyme recognition sequences), a site forligation independent cloning, a site for recombination based cloning(e.g., recombination based on att sites), a nucleotide sequencerecognized by a CRISPR/Cas (e.g. Cas9) based technology, and the like.

An insertion site can be any desirable length, and can depend on thetype of insertion site (e.g., can depend on whether (and how many) thesite includes one or more restriction enzyme recognition sequences,whether the site includes a target site for a CRISPR/Cas protein, etc.).In some cases, an insertion site of a subject nucleic acid is 3 or morenucleotides (nt) in length (e.g., 5 or more, 8 or more, 10 or more, 15or more, 17 or more, 18 or more, 19 or more, 20 or more or 25 or more,or 30 or more nt in length). In some cases, the length of an insertionsite of a subject nucleic acid has a length in a range of from 2 to 50nucleotides (nt) (e.g., from 2 to 40 nt, from 2 to 30 nt, from 2 to 25nt, from 2 to 20 nt, from 5 to 50 nt, from 5 to 40 nt, from 5 to 30 nt,from 5 to 25 nt, from 5 to 20 nt, from 10 to 50 nt, from 10 to 40 nt,from 10 to 30 nt, from 10 to 25 nt, from 10 to 20 nt, from 17 to 50 nt,from 17 to 40 nt, from 17 to 30 nt, from 17 to 25 nt). In some cases,the length of an insertion site of a subject nucleic acid has a lengthin a range of from 5 to 40 nt.

Nucleic Acid Modifications

In some embodiments, a subject nucleic acid (e.g., a Cas12J guide RNA)has one or more modifications, e.g., a base modification, a backbonemodification, etc., to provide the nucleic acid with a new or enhancedfeature (e.g., improved stability). A nucleoside is a base-sugarcombination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to the 2′, the3′, or the 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn, the respective ends ofthis linear polymeric compound can be further joined to form a circularcompound, however, linear compounds are suitable. In addition, linearcompounds may have internal nucleotide base complementarity and maytherefore fold in a manner as to produce a fully or partiallydouble-stranded compound. Within oligonucleotides, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Suitable nucleic acid modifications include, but are not limited to:2′Omethyl modified nucleotides, 2′ Fluoro modified nucleotides, lockednucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA)modified nucleotides, nucleotides with phosphorothioate linkages, and a5′ cap (e.g., a 7-methylguanylate cap (m7G)). Additional details andadditional modifications are described below.

A 2′-O-Methyl modified nucleotide (also referred to as 2′-O-Methyl RNA)is a naturally occurring modification of RNA found in tRNA and othersmall RNAs that arises as a post-transcriptional modification.Oligonucleotides can be directly synthesized that contain 2′-O-MethylRNA. This modification increases Tm of RNA:RNA duplexes but results inonly small changes in RNA:DNA stability. It is stabile with respect toattack by single-stranded ribonucleases and is typically 5 to 10-foldless susceptible to DNases than DNA. It is commonly used in antisenseoligos as a means to increase stability and binding affinity to thetarget message.

2′ Fluoro modified nucleotides (e.g., 2′ Fluoro bases) have a fluorinemodified ribose which increases binding affinity (Tm) and also conferssome relative nuclease resistance when compared to native RNA. Thesemodifications are commonly employed in ribozymes and siRNAs to improvestability in serum or other biological fluids.

LNA bases have a modification to the ribose backbone that locks the basein the C3′-endo position, which favors RNA A-type helix duplex geometry.This modification significantly increases Tm and is also very nucleaseresistant. Multiple LNA insertions can be placed in an oligo at anyposition except the 3′-end. Applications have been described rangingfrom antisense oligos to hybridization probes to SNP detection andallele specific PCR. Due to the large increase in Tm conferred by LNAs,they also can cause an increase in primer dimer formation as well asself-hairpin formation. In some cases, the number of LNAs incorporatedinto a single oligo is 10 bases or less.

The phosphorothioate (PS) bond (i.e., a phosphorothioate linkage)substitutes a sulfur atom for a non-bridging oxygen in the phosphatebackbone of a nucleic acid (e.g., an oligo). This modification rendersthe internucleotide linkage resistant to nuclease degradation.Phosphorothioate bonds can be introduced between the last 3-5nucleotides at the 5′- or 3′-end of the oligo to inhibit exonucleasedegradation. Including phosphorothioate bonds within the oligo (e.g.,throughout the entire oligo) can help reduce attack by endonucleases aswell.

In some embodiments, a subject nucleic acid has one or more nucleotidesthat are 2′-O-Methyl modified nucleotides. In some embodiments, asubject nucleic acid (e.g., a dsRNA, a siNA, etc.) has one or more 2′Fluoro modified nucleotides. In some embodiments, a subject nucleic acid(e.g., a dsRNA, a siNA, etc.) has one or more LNA bases. In someembodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) hasone or more nucleotides that are linked by a phosphorothioate bond(i.e., the subject nucleic acid has one or more phosphorothioatelinkages). In some embodiments, a subject nucleic acid (e.g., a dsRNA, asiNA, etc.) has a 5′ cap (e.g., a 7-methylguanylate cap (m7G)). In someembodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) has acombination of modified nucleotides. For example, a subject nucleic acid(e.g., a dsRNA, a siNA, etc.) can have a 5′ cap (e.g., a7-methylguanylate cap (m7G)) in addition to having one or morenucleotides with other modifications (e.g., a 2′-O-Methyl nucleotideand/or a 2′ Fluoro modified nucleotide and/or a LNA base and/or aphosphorothioate linkage).

Modified Backbones and Modified Internucleoside Linkages

Examples of suitable nucleic acids (e.g., a Cas12J guide RNA) containingmodifications include nucleic acids containing modified backbones ornon-natural internucleoside linkages. Nucleic acids having modifiedbackbones include those that retain a phosphorus atom in the backboneand those that do not have a phosphorus atom in the backbone.

Suitable modified oligonucleotide backbones containing a phosphorus atomtherein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be a basic (the nucleobase is missing or has ahydroxyl group in place thereof). Various salts (such as, for example,potassium or sodium), mixed salts and free acid forms are also included.

In some embodiments, a subject nucleic acid comprises one or morephosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). MMI type internucleoside linkages are disclosed inthe above referenced U.S. Pat. No. 5,489,677, the disclosure of which isincorporated herein by reference in its entirety. Suitable amideinternucleoside linkages are disclosed in U.S. Pat. No. 5,602,240, thedisclosure of which is incorporated herein by reference in its entirety.

Also suitable are nucleic acids having morpholino backbone structures asdescribed in, e.g., U.S. Pat. No. 5,034,506. For example, in someembodiments, a subject nucleic acid comprises a 6-membered morpholinoring in place of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagereplaces a phosphodiester linkage.

Suitable modified polynucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; fonnacetyl and thiofonnacetyl backbones; methylene fonnacetyland thiofonnacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Mimetics

A subject nucleic acid can be a nucleic acid mimetic. The term “mimetic”as it is applied to polynucleotides is intended to includepolynucleotides wherein only the furanose ring or both the furanose ringand the internucleotide linkage are replaced with non-furanose groups,replacement of only the furanose ring is also referred to in the art asbeing a sugar surrogate. The heterocyclic base moiety or a modifiedheterocyclic base moiety is maintained for hybridization with anappropriate target nucleic acid. One such nucleic acid, a polynucleotidemimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA, thesugar-backbone of a polynucleotide is replaced with an amide containingbackbone, in particular an aminoethylglycine backbone. The nucleotidesare retained and are bound directly or indirectly to aza nitrogen atomsof the amide portion of the backbone.

One polynucleotide mimetic that has been reported to have excellenthybridization properties is a peptide nucleic acid (PNA). The backbonein PNA compounds is two or more linked aminoethylglycine units whichgives PNA an amide containing backbone. The heterocyclic base moietiesare bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that describe thepreparation of PNA compounds include, but are not limited to: U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, the disclosures of which areincorporated herein by reference in their entirety.

Another class of polynucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. One class of linking groups has been selected to give anon-ionic oligomeric compound. The non-ionic morpholino-based oligomericcompounds are less likely to have undesired interactions with cellularproteins. Morpholino-based polynucleotides are non-ionic mimics ofoligonucleotides which are less likely to form undesired interactionswith cellular proteins (Dwaine A. Braasch and David R. Corey,Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotidesare disclosed in U.S. Pat. No. 5,034,506, the disclosure of which isincorporated herein by reference in its entirety. A variety of compoundswithin the morpholino class of polynucleotides have been prepared,having a variety of different linking groups joining the monomericsubunits.

A further class of polynucleotide mimetic is referred to as cyclohexenylnucleic acids (CeNA). The furanose ring normally present in a DNA/RNAmolecule is replaced with a cyclohexenyl ring. CeNA DMT protectedphosphoramidite monomers have been prepared and used for oligomericcompound synthesis following classical phosphoramidite chemistry. Fullymodified CeNA oligomeric compounds and oligonucleotides having specificpositions modified with CeNA have been prepared and studied (see Wang etal., J. Am. Chem. Soc., 2000, 122, 8595-8602, the disclosure of which isincorporated herein by reference in its entirety). In general theincorporation of CeNA monomers into a DNA chain increases its stabilityof a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA andDNA complements with similar stability to the native complexes. Thestudy of incorporating CeNA structures into natural nucleic acidstructures was shown by NMR and circular dichroism to proceed with easyconformational adaptation.

A further modification includes Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ringthereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety. The linkage can be a methylene (—CH₂—), groupbridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2(Singh et al., Chem. Commun., 1998, 4, 455-456, the disclosure of whichis incorporated herein by reference in its entirety). LNA and LNAanalogs display very high duplex thermal stabilities with complementaryDNA and RNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolyticdegradation and good solubility properties. Potent and nontoxicantisense oligonucleotides containing LNAs have been described (e.g.,Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638,the disclosure of which is incorporated herein by reference in itsentirety).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methylcytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630, thedisclosure of which is incorporated herein by reference in itsentirety). LNAs and preparation thereof are also described in WO98/39352 and WO 99/14226, as well as U.S. applications 20120165514,20100216983, 20090041809, 20060117410, 20040014959, 20020094555, and20020086998, the disclosures of which are incorporated herein byreference in their entirety.

Modified Sugar Moieties

A subject nucleic acid can also include one or more substituted sugarmoieties. Suitable polynucleotides comprise a sugar substituent groupselected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C.sub.1 to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly suitable are O((CH₂)_(n)O)_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Othersuitable polynucleotides comprise a sugar substituent group selectedfrom: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Asuitable modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504, the disclosure of which is incorporated hereinby reference in its entirety) i.e., an alkoxyalkoxy group. A furthersuitable modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups include methoxy (—O—CH₃),aminopropoxy CH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl CH₂—CH═CH₂)and fluoro (F). 2′-sugar substituent groups may be in the arabino (up)position or ribo (down) position. A suitable 2′-arabino modification is2′-F. Similar modifications may also be made at other positions on theoligomeric compound, particularly the 3′ position of the sugar on the 3′terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligomeric compounds may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar.

Base Modifications and Substitutions

A subject nucleic acid may also include nucleobase (often referred to inthe art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993; the disclosures ofwhich are incorporated herein by reference in their entirety. Certain ofthese nucleobases are useful for increasing the binding affinity of anoligomeric compound. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi et al., eds., AntisenseResearch and Applications, CRC Press, Boca Raton, 1993, pp. 276-278; thedisclosure of which is incorporated herein by reference in its entirety)and are suitable base substitutions, e.g., when combined with2′-O-methoxyethyl sugar modifications.

Conjugates

Another possible modification of a subject nucleic acid involveschemically linking to the polynucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups include, but are notlimited to, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Suitable conjugate groupsinclude, but are not limited to, cholesterols, lipids, phospholipids,biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties include groups that improve uptake, enhanceresistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties include groups that improve uptake,distribution, metabolism or excretion of a subject nucleic acid.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

A conjugate may include a “Protein Transduction Domain” or PTD (alsoknown as a CPP—cell penetrating peptide), which may refer to apolypeptide, polynucleotide, carbohydrate, or organic or inorganiccompound that facilitates traversing a lipid bilayer, micelle, cellmembrane, organelle membrane, or vesicle membrane. A PTD attached toanother molecule, which can range from a small polar molecule to a largemacromolecule and/or a nanoparticle, facilitates the molecule traversinga membrane, for example going from extracellular space to intracellularspace, or cytosol to within an organelle (e.g., the nucleus). In someembodiments, a PTD is covalently linked to the 3′ end of an exogenouspolynucleotide. In some embodiments, a PTD is covalently linked to the5′ end of an exogenous polynucleotide. Exemplary PTDs include but arenot limited to a minimal undecapeptide protein transduction domain(corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR;SEQ ID NO: 64); a polyarginine sequence comprising a number of argininessufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10,or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer GeneTher. 9(6):489-96); an Drosophila Antennapedia protein transductiondomain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncatedhuman calcitonin peptide (Trehin et al. (2004) Pharm. Research21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci.USA 97:13003-13008); RRQRRTSKLMKR SEQ ID NO: 65); TransportanGWTLNSAGYLLGKINLKALAALAKKIL SEQ ID NO: 66);KALAWEAKLAKALAKALAKHLAKALAKALKCEA SEQ ID NO: 67); and RQIKIWFQNRRMKWKKSEQ ID NO: 68). Exemplary PTDs include but are not limited to,YGRKKRRQRRR SEQ ID NO: 64), RKKRRQRRR SEQ ID NO: 69); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;Exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following: YGRKKRRQRRR SEQ ID NO: 64); RKKRRQRR SEQ IDNO: 69); YARAAARQARA SEQ ID NO: 71); THRLPRRRRRR SEQ ID NO: 72); andGGRRARRRRRR SEQ ID NO: 73). In some embodiments, the PTD is anactivatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”)connected via a cleavable linker to a matching polyanion (e.g., Glu9 or“E9”), which reduces the net charge to nearly zero and thereby inhibitsadhesion and uptake into cells. Upon cleavage of the linker, thepolyanion is released, locally unmasking the polyarginine and itsinherent adhesiveness, thus “activating” the ACPP to traverse themembrane.

Introducing Components into a Target Cell

A Cas12J guide RNA (or a nucleic acid comprising a nucleotide sequenceencoding same) and/or a Cas12J polypeptide of the present disclosure (ora nucleic acid comprising a nucleotide sequence encoding same) and/or aCas12J fusion polypeptide of the present disclosure (or a nucleic acidthat includes a nucleotide sequence encoding a Cas12J fusion polypeptideof the present disclosure) and/or a donor polynucleotide (donortemplate) can be introduced into a host cell by any of a variety ofwell-known methods.

Any of a variety of compounds and methods can be used to deliver to atarget cell a Cas12J system of the present disclosure (e.g., where aCas12J system comprises: a) a Cas12J polypeptide of the presentdisclosure and a Cas12J guide RNA; b) a Cas12J polypeptide of thepresent disclosure, a Cas12J guide RNA, and a donor template nucleicacid; c) a Cas12J fusion polypeptide of the present disclosure and aCas12J guide RNA; d) a Cas12J fusion polypeptide of the presentdisclosure, a Cas12J guide RNA, and a donor template nucleic acid; e) anmRNA encoding a Cas12J polypeptide of the present disclosure; and aCas12J guide RNA; f) an mRNA encoding a Cas12J polypeptide of thepresent disclosure, a Cas12J guide RNA, and a donor template nucleicacid; g) an mRNA encoding a Cas12J fusion polypeptide of the presentdisclosure; and a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusionpolypeptide of the present disclosure, a Cas12J guide RNA, and a donortemplate nucleic acid; i) a recombinant expression vector comprising anucleotide sequence encoding a Cas12J polypeptide of the presentdisclosure and a nucleotide sequence encoding a Cas12J guide RNA; j) arecombinant expression vector comprising a nucleotide sequence encodinga Cas12J polypeptide of the present disclosure, a nucleotide sequenceencoding a Cas12J guide RNA, and a nucleotide sequence encoding a donortemplate nucleic acid; k) a recombinant expression vector comprising anucleotide sequence encoding a Cas12J fusion polypeptide of the presentdisclosure and a nucleotide sequence encoding a Cas12J guide RNA; 1) arecombinant expression vector comprising a nucleotide sequence encodinga Cas12J fusion polypeptide of the present disclosure, a nucleotidesequence encoding a Cas12J guide RNA, and a nucleotide sequence encodinga donor template nucleic acid; m) a first recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure, and a second recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J guide RNA; n) a firstrecombinant expression vector comprising a nucleotide sequence encodinga Cas12J polypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jguide RNA; and a donor template nucleic acid; o) a first recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jfusion polypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jguide RNA; p) a first recombinant expression vector comprising anucleotide sequence encoding a Cas12J fusion polypeptide of the presentdisclosure, and a second recombinant expression vector comprising anucleotide sequence encoding a Cas12J guide RNA; and a donor templatenucleic acid; q) a recombinant expression vector comprising a nucleotidesequence encoding a Cas12J polypeptide of the present disclosure, anucleotide sequence encoding a first Cas12J guide RNA, and a nucleotidesequence encoding a second Cas12J guide RNA; or r) a recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jfusion polypeptide of the present disclosure, a nucleotide sequenceencoding a first Cas12J guide RNA, and a nucleotide sequence encoding asecond Cas12J guide RNA; or some variation of one of (a) through (r). Asa non-limiting example, a Cas12J system of the present disclosure can becombined with a lipid. As another non-limiting example, a Cas12J systemof the present disclosure can be combined with a particle, or formulatedinto a particle.

Methods of introducing a nucleic acid into a host cell are known in theart, and any convenient method can be used to introduce a subjectnucleic acid (e.g., an expression construct/vector) into a target cell(e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell,mammalian cell, human cell, and the like). Suitable methods include,e.g., viral infection, transfection, conjugation, protoplast fusion,lipofection, electroporation, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro injection,nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., alAdv Drug Deliv Rev. 2012 Sep. 13. pii: 50169-409X(12)00283-9. doi:10.1016/j.addr.2012.09.023), and the like.

In some cases, a Cas12J polypeptide of the present disclosure isprovided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid, anexpression vector, a viral vector, etc.) that encodes the Cas12Jpolypeptide. In some cases, the Cas12J polypeptide of the presentdisclosure is provided directly as a protein (e.g., without anassociated guide RNA or with an associate guide RNA, i.e., as aribonucleoprotein complex). A Cas12J polypeptide of the presentdisclosure can be introduced into a cell (provided to the cell) by anyconvenient method; such methods are known to those of ordinary skill inthe art. As an illustrative example, a Cas12J polypeptide of the presentdisclosure can be injected directly into a cell (e.g., with or without aCas12J guide RNA or nucleic acid encoding a Cas12J guide RNA, and withor without a donor polynucleotide). As another example, a preformedcomplex of a Cas12J polypeptide of the present disclosure and a Cas12Jguide RNA (an RNP) can be introduced into a cell (e.g, eukaryotic cell)(e.g., via injection, via nucleofection; via a protein transductiondomain (PTD) conjugated to one or more components, e.g., conjugated tothe Cas12J protein, conjugated to a guide RNA, conjugated to a Cas12Jpolypeptide of the present disclosure and a guide RNA; etc.).

In some cases, a Cas12J fusion polypeptide (e.g., dCas12J fused to afusion partner, nickase Cas12J fused to a fusion partner, etc.) of thepresent disclosure is provided as a nucleic acid (e.g., an mRNA, a DNA,a plasmid, an expression vector, a viral vector, etc.) that encodes theCas12J fusion polypeptide. In some cases, the Cas12J fusion polypeptideof the present disclosure is provided directly as a protein (e.g.,without an associated guide RNA or with an associate guide RNA, i.e., asa ribonucleoprotein complex). A Cas12J fusion polypeptide of the presentdisclosure can be introduced into a cell (provided to the cell) by anyconvenient method; such methods are known to those of ordinary skill inthe art. As an illustrative example, a Cas12J fusion polypeptide of thepresent disclosure can be injected directly into a cell (e.g., with orwithout nucleic acid encoding a Cas12J guide RNA and with or without adonor polynucleotide). As another example, a preformed complex of aCas12J fusion polypeptide of the present disclosure and a Cas12J guideRNA (an RNP) can be introduced into a cell (e.g., via injection, vianucleofection; via a protein transduction domain (PTD) conjugated to oneor more components, e.g., conjugated to the Cas12J fusion protein,conjugated to a guide RNA, conjugated to a Cas12J fusion polypeptide ofthe present disclosure and a guide RNA; etc.).

In some cases, a nucleic acid (e.g., a Cas12J guide RNA; a nucleic acidcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure; etc.) is delivered to a cell (e.g., a target hostcell) and/or a polypeptide (e.g., a Cas12J polypeptide; a Cas12J fusionpolypeptide) in a particle, or associated with a particle. In somecases, a Cas12J system of the present disclosure is delivered to a cellin a particle, or associated with a particle. The terms “particle” andnanoparticle” can be used interchangeable, as appropriate. A recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jpolypeptide of the present disclosure and/or a Cas12J guide RNA, an mRNAcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure, and guide RNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, a Cas12J polypeptide and aCas12J guide RNA, e.g., as a complex (e.g., a ribonucleoprotein (RNP)complex), can be delivered via a particle, e.g., a delivery particlecomprising lipid or lipidoid and hydrophilic polymer, e.g., a cationiclipid and a hydrophilic polymer, for instance wherein the cationic lipidcomprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5). Forexample, a particle can be formed using a multistep process in which aCas12J polypeptide and a Cas12J guideRNA are mixed together, e.g., at a1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g.,in sterile, nuclease free 1× phosphate-buffered saline (PBS); andseparately, DOTAP, DMPC, PEG, and cholesterol as applicable for theformulation are dissolved in alcohol, e.g., 100% ethanol; and, the twosolutions are mixed together to form particles containing thecomplexes).

A Cas12J polypeptide of the present disclosure (or an mRNA comprising anucleotide sequence encoding a Cas12J polypeptide of the presentdisclosure; or a recombinant expression vector comprising a nucleotidesequence encoding a Cas12J polypeptide of the present disclosure) and/orCas12J guide RNA (or a nucleic acid such as one or more expressionvectors encoding the Cas12J guide RNA) may be delivered simultaneouslyusing particles or lipid envelopes. For example, a biodegradablecore-shell structured nanoparticle with a poly ((3-amino ester) (PBAE)core enveloped by a phospholipid bilayer shell can be used. In somecases, particles/nanoparticles based on self assembling bioadhesivepolymers are used; such particles/nanoparticles may be applied to oraldelivery of peptides, intravenous delivery of peptides and nasaldelivery of peptides, e.g., to the brain. Other embodiments, such asoral absorption and ocular delivery of hydrophobic drugs are alsocontemplated. A molecular envelope technology, which involves anengineered polymer envelope which is protected and delivered to the siteof the disease, can be used. Doses of about 5 mg/kg can be used, withsingle or multiple doses, depending on various factors, e.g., the targettissue.

Lipidoid compounds (e.g., as described in US patent application20110293703) are also useful in the administration of polynucleotides,and can be used to deliver a Cas12J polypeptide of the presentdisclosure, a Cas12J fusion polypeptide of the present disclosure, anRNP of the present disclosure, a nucleic acid of the present disclosure,or a Cas12J system of the present disclosure (e.g., where a Cas12Jsystem comprises: a) a Cas12J polypeptide of the present disclosure anda Cas12J guide RNA; b) a Cas12J polypeptide of the present disclosure, aCas12J guide RNA, and a donor template nucleic acid; c) a Cas12J fusionpolypeptide of the present disclosure and a Cas12J guide RNA; d) aCas12J fusion polypeptide of the present disclosure, a Cas12J guide RNA,and a donor template nucleic acid; e) an mRNA encoding a Cas12Jpolypeptide of the present disclosure; and a Cas12J guide RNA; f) anmRNA encoding a Cas12J polypeptide of the present disclosure, a Cas12Jguide RNA, and a donor template nucleic acid; g) an mRNA encoding aCas12J fusion polypeptide of the present disclosure; and a Cas12J guideRNA; h) an mRNA encoding a Cas12J fusion polypeptide of the presentdisclosure, a Cas12J guide RNA, and a donor template nucleic acid; i) arecombinant expression vector comprising a nucleotide sequence encodinga Cas12J polypeptide of the present disclosure and a nucleotide sequenceencoding a Cas12J guide RNA; j) a recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure, a nucleotide sequence encoding a Cas12J guide RNA,and a nucleotide sequence encoding a donor template nucleic acid; k) arecombinant expression vector comprising a nucleotide sequence encodinga Cas12J fusion polypeptide of the present disclosure and a nucleotidesequence encoding a Cas12J guide RNA; 1) a recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J fusion polypeptide ofthe present disclosure, a nucleotide sequence encoding a Cas12J guideRNA, and a nucleotide sequence encoding a donor template nucleic acid;m) a first recombinant expression vector comprising a nucleotidesequence encoding a Cas12J polypeptide of the present disclosure, and asecond recombinant expression vector comprising a nucleotide sequenceencoding a Cas12J guide RNA; n) a first recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure, and a second recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J guide RNA; and adonor template nucleic acid; o) a first recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J fusion polypeptide ofthe present disclosure, and a second recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J guide RNA; p) a firstrecombinant expression vector comprising a nucleotide sequence encodinga Cas12J fusion polypeptide of the present disclosure, and a secondrecombinant expression vector comprising a nucleotide sequence encodinga Cas12J guide RNA; and a donor template nucleic acid; q) a recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jpolypeptide of the present disclosure, a nucleotide sequence encoding afirst Cas12J guide RNA, and a nucleotide sequence encoding a secondCas12J guide RNA; or r) a recombinant expression vector comprising anucleotide sequence encoding a Cas12J fusion polypeptide of the presentdisclosure, a nucleotide sequence encoding a first Cas12J guide RNA, anda nucleotide sequence encoding a second Cas12J guide RNA; or somevariation of one of (a) through (r). In one aspect, the aminoalcohollipidoid compounds are combined with an agent to be delivered to a cellor a subject to form microparticles, nanoparticles, liposomes, ormicelles. The aminoalcohol lipidoid compounds may be combined with otheraminoalcohol lipidoid compounds, polymers (synthetic or natural),surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to formthe particles. These particles may then optionally be combined with apharmaceutical excipient to form a pharmaceutical composition.

A poly(beta-amino alcohol) (PBAA) can be used to deliver a Cas12Jpolypeptide of the present disclosure, a Cas12J fusion polypeptide ofthe present disclosure, an RNP of the present disclosure, a nucleic acidof the present disclosure, or a Cas12J system of the present disclosure,to a target cell. US Patent Publication No. 20130302401 relates to aclass of poly(beta-amino alcohols) (PBAAs) that has been prepared usingcombinatorial polymerization.

Sugar-based particles may be used, for example GalNAc, as described withreference to WO2014118272 (incorporated herein by reference) and Nair, JK et al., 2014, Journal of the American Chemical Society 136 (49),16958-16961) can be used to deliver a Cas12J polypeptide of the presentdisclosure, a Cas12J fusion polypeptide of the present disclosure, anRNP of the present disclosure, a nucleic acid of the present disclosure,or a Cas12J system of the present disclosure, to a target cell.

In some cases, lipid nanoparticles (LNPs) are used to deliver a Cas12Jpolypeptide of the present disclosure, a Cas12J fusion polypeptide ofthe present disclosure, an RNP of the present disclosure, a nucleic acidof the present disclosure, or a Cas12J system of the present disclosure,to a target cell. Negatively charged polymers such as RNA may be loadedinto LNPs at low pH values (e.g., pH 4) where the ionizable lipidsdisplay a positive charge. However, at physiological pH values, the LNPsexhibit a low surface charge compatible with longer circulation times.Four species of ionizable cationic lipids have been focused upon, namely1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).Preparation of LNPs and is described in, e.g., Rosin et al. (2011)Molecular Therapy 19:1286-2200). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(.omega.-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be used. Anucleic acid (e.g., a Cas12J guide RNA; a nucleic acid of the presentdisclosure; etc.) may be encapsulated in LNPs containing DLinDAP,DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMGor PEG-C-DOMG at 40:10:40:10 molar ratios). In some cases, 0.2%SP-DiOC18 is incorporated.

Spherical Nucleic Acid (SNA™) constructs and other nanoparticles(particularly gold nanoparticles) can be used to deliver a Cas12Jpolypeptide of the present disclosure, a Cas12J fusion polypeptide ofthe present disclosure, an RNP of the present disclosure, a nucleic acidof the present disclosure, or a Cas12J system of the present disclosure,to a target cell. See, e.g., Cutler et al., J. Am. Chem. Soc. 2011133:9254-9257, Hao et al., Small 2011 7:3158-3162, Zhang et al., ACSNano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al.,Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 20127:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691,Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci.USA. 2013 110(19): 7625-7630, Jensen et al., Sci. Transl. Med. 5,209ra152 (2013) and Mirkin, et al., Small, 10:186-192.

Self-assembling nanoparticles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG).

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In some cases, nanoparticles suitable for use indelivering a Cas12J polypeptide of the present disclosure, a Cas12Jfusion polypeptide of the present disclosure, an RNP of the presentdisclosure, a nucleic acid of the present disclosure, or a Cas12J systemof the present disclosure, to a target cell have a diameter of 500 nm orless, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm,from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm.In some cases, nanoparticles suitable for use in delivering a Cas12Jpolypeptide of the present disclosure, a Cas12J fusion polypeptide ofthe present disclosure, an RNP of the present disclosure, a nucleic acidof the present disclosure, or a Cas12J system of the present disclosure,to a target cell have a diameter of from 25 nm to 200 nm. In some cases,nanoparticles suitable for use in delivering a Cas12J polypeptide of thepresent disclosure, a Cas12J fusion polypeptide of the presentdisclosure, an RNP of the present disclosure, a nucleic acid of thepresent disclosure, or a Cas12J system of the present disclosure, to atarget cell have a diameter of 100 nm or less In some cases,nanoparticles suitable for use in delivering a Cas12J polypeptide of thepresent disclosure, a Cas12J fusion polypeptide of the presentdisclosure, an RNP of the present disclosure, a nucleic acid of thepresent disclosure, or a Cas12J system of the present disclosure, to atarget cell have a diameter of from 35 nm to 60 nm.

Nanoparticles suitable for use in delivering a Cas12J polypeptide of thepresent disclosure, a Cas12J fusion polypeptide of the presentdisclosure, an RNP of the present disclosure, a nucleic acid of thepresent disclosure, or a Cas12J system of the present disclosure, to atarget cell may be provided in different forms, e.g., as solidnanoparticles (e.g., metal such as silver, gold, iron, titanium),non-metal, lipid-based solids, polymers), suspensions of nanoparticles,or combinations thereof. Metal, dielectric, and semiconductornanoparticles may be prepared, as well as hybrid structures (e.g.,core-shell nanoparticles). Nanoparticles made of semiconducting materialmay also be labeled quantum dots if they are small enough (typicallybelow 10 nm) that quantization of electronic energy levels occurs. Suchnanoscale particles are used in biomedical applications as drug carriersor imaging agents and may be adapted for similar purposes in the presentdisclosure.

Semi-solid and soft nanoparticles are also suitable for use indelivering a Cas12J polypeptide of the present disclosure, a Cas12Jfusion polypeptide of the present disclosure, an RNP of the presentdisclosure, a nucleic acid of the present disclosure, or a Cas12J systemof the present disclosure, to a target cell. A prototype nanoparticle ofsemi-solid nature is the liposome.

In some cases, an exosome is used to deliver a Cas12J polypeptide of thepresent disclosure, a Cas12J fusion polypeptide of the presentdisclosure, an RNP of the present disclosure, a nucleic acid of thepresent disclosure, or a Cas12J system of the present disclosure, to atarget cell. Exosomes are endogenous nano-vesicles that transport RNAsand proteins, and which can deliver RNA to the brain and other targetorgans.

In some cases, a liposome is used to deliver a Cas12J polypeptide of thepresent disclosure, a Cas12J fusion polypeptide of the presentdisclosure, an RNP of the present disclosure, a nucleic acid of thepresent disclosure, or a Cas12J system of the present disclosure, to atarget cell. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes can be made from several different types of lipids;however, phospholipids are most commonly used to generate liposomes.Although liposome formation is spontaneous when a lipid film is mixedwith an aqueous solution, it can also be expedited by applying force inthe form of shaking by using a homogenizer, sonicator, or an extrusionapparatus. Several other additives may be added to liposomes in order tomodify their structure and properties. For instance, either cholesterolor sphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. A liposome formulation may be mainly comprised ofnatural phospholipids and lipids such as1,2-distearoyl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin,egg phosphatidylcholines and monosialoganglioside.

A stable nucleic-acid-lipid particle (SNALP) can be used to deliver aCas12J polypeptide of the present disclosure, a Cas12J fusionpolypeptide of the present disclosure, an RNP of the present disclosure,a nucleic acid of the present disclosure, or a Cas12J system of thepresent disclosure, to a target cell. The SNALP formulation may containthe lipids 3-N-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio. The SNALP liposomes may be prepared byformulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine(DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. Theresulting SNALP liposomes can be about 80-100 nm in size. A SNALP maycomprise synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA),dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala.,USA), 3-N-[(w-methoxy poly(ethyleneglycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic1,2-dilinoleyloxy-3-N,Ndimethylaminopropane. A SNALP may comprisesynthetic cholesterol (Sigma-Aldrich),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar LipidsInc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane(DLinDMA).

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) canbe used to deliver a Cas12J polypeptide of the present disclosure, aCas12J fusion polypeptide of the present disclosure, an RNP of thepresent disclosure, a nucleic acid of the present disclosure, or aCas12J system of the present disclosure, to a target cell. A preformedvesicle with the following lipid composition may be contemplated: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11.+−0.0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Lipids may be formulated with a Cas12J system of the present disclosureor component(s) thereof or nucleic acids encoding the same to form lipidnanoparticles (LNPs). Suitable lipids include, but are not limited to,DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline,cholesterol, and PEG-DMG may be formulated with a Cas12J system, orcomponent thereof, of the present disclosure, using a spontaneousvesicle formation procedure. The component molar ratio may be about50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG).

A Cas12J system of the present disclosure, or a component thereof, maybe delivered encapsulated in PLGA microspheres such as that furtherdescribed in US published applications 20130252281 and 20130245107 and20130244279.

Supercharged proteins can be used to deliver a Cas12J polypeptide of thepresent disclosure, a Cas12J fusion polypeptide of the presentdisclosure, an RNP of the present disclosure, a nucleic acid of thepresent disclosure, or a Cas12J system of the present disclosure, to atarget cell. Supercharged proteins are a class of engineered ornaturally occurring proteins with unusually high positive or negativenet theoretical charge. Both supernegatively and superpositively chargedproteins exhibit the ability to withstand thermally or chemicallyinduced aggregation. Superpositively charged proteins are also able topenetrate mammalian cells. Associating cargo with these proteins, suchas plasmid DNA, RNA, or other proteins, can enable the functionaldelivery of these macromolecules into mammalian cells both in vitro andin vivo.

Cell Penetrating Peptides (CPPs) can be used to deliver a Cas12Jpolypeptide of the present disclosure, a Cas12J fusion polypeptide ofthe present disclosure, an RNP of the present disclosure, a nucleic acidof the present disclosure, or a Cas12J system of the present disclosure,to a target cell. CPPs typically have an amino acid composition thateither contains a high relative abundance of positively charged aminoacids such as lysine or arginine or has sequences that contain analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids.

An implantable device can be used to deliver a Cas12J polypeptide of thepresent disclosure, a Cas12J fusion polypeptide of the presentdisclosure, an RNP of the present disclosure, a nucleic acid of thepresent disclosure (e.g., a Cas12J guide RNA, a nucleic acid encoding aCas12J guide RNA, a nucleic acid encoding Cas12J polypeptide, a donortemplate, and the like), or a Cas12J system of the present disclosure,to a target cell (e.g., a target cell in vivo, where the target cell isa target cell in circulation, a target cell in a tissue, a target cellin an organ, etc.). An implantable device suitable for use in deliveringa Cas12J polypeptide of the present disclosure, a Cas12J fusionpolypeptide of the present disclosure, an RNP of the present disclosure,a nucleic acid of the present disclosure, or a Cas12J system of thepresent disclosure, to a target cell (e.g., a target cell in vivo, wherethe target cell is a target cell in circulation, a target cell in atissue, a target cell in an organ, etc.) can include a container (e.g.,a reservoir, a matrix, etc.) that comprises the Cas12J polypeptide, theCas12J fusion polypeptide, the RNP, or the Cas12J system (or componentthereof, e.g., a nucleic acid of the present disclosure).

A suitable implantable device can comprise a polymeric substrate, suchas a matrix for example, that is used as the device body, and in somecases additional scaffolding materials, such as metals or additionalpolymers, and materials to enhance visibility and imaging. Animplantable delivery device can be advantageous in providing releaselocally and over a prolonged period, where the polypeptide and/ornucleic acid to be delivered is released directly to a target site,e.g., the extracellular matrix (ECM), the vasculature surrounding atumor, a diseased tissue, etc. Suitable implantable delivery devicesinclude devices suitable for use in delivering to a cavity such as theabdominal cavity and/or any other type of administration in which thedrug delivery system is not anchored or attached, comprising a biostableand/or degradable and/or bioabsorbable polymeric substrate, which mayfor example optionally be a matrix. In some cases, a suitableimplantable drug delivery device comprises degradable polymers, whereinthe main release mechanism is bulk erosion. In some cases, a suitableimplantable drug delivery device comprises non degradable, or slowlydegraded polymers, wherein the main release mechanism is diffusionrather than bulk erosion, so that the outer part functions as membrane,and its internal part functions as a drug reservoir, which practicallyis not affected by the surroundings for an extended period (for examplefrom about a week to about a few months). Combinations of differentpolymers with different release mechanisms may also optionally be used.The concentration gradient at the can be maintained effectively constantduring a significant period of the total releasing period, and thereforethe diffusion rate is effectively constant (termed “zero mode”diffusion). By the term “constant” it is meant a diffusion rate that ismaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate can be so maintained for a prolonged period, and itcan be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

In some cases, the implantable delivery system is designed to shield thenucleotide based therapeutic agent from degradation, whether chemical innature or due to attack from enzymes and other factors in the body ofthe subject.

The site for implantation of the device, or target site, can be selectedfor maximum therapeutic efficacy. For example, a delivery device can beimplanted within or in the proximity of a tumor environment, or theblood supply associated with a tumor. The target location can be,e.g.: 1) the brain at degenerative sites like in Parkinson or Alzheimerdisease at the basal ganglia, white and gray matter; 2) the spine, as inthe case of amyotrophic lateral sclerosis (ALS); 3) uterine cervix; 4)active and chronic inflammatory joints; 5) dermis as in the case ofpsoriasis; 7) sympathetic and sensoric nervous sites for analgesiceffect; 7) a bone; 8) a site of acute or chronic infection; 9) Intravaginal; 10) Inner ear-auditory system, labyrinth of the inner ear,vestibular system; 11) Intra tracheal; 12) Intra-cardiac; coronary,epicardiac; 13) urinary tract or bladder; 14) biliary system; 15)parenchymal tissue including and not limited to the kidney, liver,spleen; 16) lymph nodes; 17) salivary glands; 18) dental gums; 19)Intra-articular (into joints); 20) Intra-ocular; 21) Brain tissue; 22)Brain ventricles; 23) Cavities, including abdominal cavity (for examplebut without limitation, for ovary cancer); 24) Intra esophageal; and 25)Intra rectal; and 26) into the vasculature.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as stereotacticmethods into the brain tissue, laparoscopy, including implantation witha laparoscope into joints, abdominal organs, the bladder wall and bodycavities.

Modified Host Cells

The present disclosure provides a modified cell comprising a Cas12Jpolypeptide of the present disclosure and/or a nucleic acid comprising anucleotide sequence encoding a Cas12J polypeptide of the presentdisclosure. The present disclosure provides a modified cell comprising aCas12J polypeptide of the present disclosure, where the modified cell isa cell that does not normally comprise a Cas12J polypeptide of thepresent disclosure. The present disclosure provides a modified cell(e.g., a genetically modified cell) comprising nucleic acid comprising anucleotide sequence encoding a Cas12J polypeptide of the presentdisclosure. The present disclosure provides a genetically modified cellthat is genetically modified with an mRNA comprising a nucleotidesequence encoding a Cas12J polypeptide of the present disclosure. Thepresent disclosure provides a genetically modified cell that isgenetically modified with a recombinant expression vector comprising anucleotide sequence encoding a Cas12J polypeptide of the presentdisclosure. The present disclosure provides a genetically modified cellthat is genetically modified with a recombinant expression vectorcomprising: a) a nucleotide sequence encoding a Cas12J polypeptide ofthe present disclosure; and b) a nucleotide sequence encoding a Cas12Jguide RNA of the present disclosure. The present disclosure provides agenetically modified cell that is genetically modified with arecombinant expression vector comprising: a) a nucleotide sequenceencoding a Cas12J polypeptide of the present disclosure; b) a nucleotidesequence encoding a Cas12J guide RNA of the present disclosure; and c) anucleotide sequence encoding a donor template.

A cell that serves as a recipient for a Cas12J polypeptide of thepresent disclosure and/or a nucleic acid comprising a nucleotidesequence encoding a Cas12J polypeptide of the present disclosure and/ora Cas12J guide RNA of the present disclosure, can be any of a variety ofcells, including, e.g., in vitro cells; in vivo cells; ex vivo cells;primary cells; cancer cells; animal cells; plant cells; algal cells;fungal cells; etc. A cell that serves as a recipient for a Cas12Jpolypeptide of the present disclosure and/or a nucleic acid comprising anucleotide sequence encoding a Cas12J polypeptide of the presentdisclosure and/or a Cas12J guide RNA of the present disclosure isreferred to as a “host cell” or a “target cell.” A host cell or a targetcell can be a recipient of a Cas12J system of the present disclosure. Ahost cell or a target cell can be a recipient of a Cas12J RNP of thepresent disclosure. A host cell or a target cell can be a recipient of asingle component of a Cas12J system of the present disclosure.

Non-limiting examples of cells (target cells) include: a prokaryoticcell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of asingle-cell eukaryotic organism, a protozoa cell, a cell from a plant(e.g., cells from plant crops, fruits, vegetables, grains, soy bean,corn, maize, wheat, seeds, tomatos, rice, cassava, sugarcane, pumpkin,hay, potatos, cotton, Cannabis, tobacco, flowering plants, conifers,gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts,mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g.,Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsisgaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and thelike), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cellfrom a mushroom), an animal cell, a cell from an invertebrate animal(e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from avertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cellfrom a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep);a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline(e.g., a cat); a canine (e.g., a dog); etc.), and the like. In somecases, the cell is a cell that does not originate from a naturalorganism (e.g., the cell can be a synthetically made cell; also referredto as an artificial cell).

A cell can be an in vitro cell (e.g., established cultured cell line). Acell can be an ex vivo cell (cultured cell from an individual). A cellcan be and in vivo cell (e.g., a cell in an individual). A cell can bean isolated cell. A cell can be a cell inside of an organism. A cell canbe an organism. A cell can be a cell in a cell culture (e.g., in vitrocell culture). A cell can be one of a collection of cells. A cell can bea prokaryotic cell or derived from a prokaryotic cell. A cell can be abacterial cell or can be derived from a bacterial cell. A cell can be anarchaeal cell or derived from an archaeal cell. A cell can be aeukaryotic cell or derived from a eukaryotic cell. A cell can be a plantcell or derived from a plant cell. A cell can be an animal cell orderived from an animal cell. A cell can be an invertebrate cell orderived from an invertebrate cell. A cell can be a vertebrate cell orderived from a vertebrate cell. A cell can be a mammalian cell orderived from a mammalian cell. A cell can be a rodent cell or derivedfrom a rodent cell. A cell can be a human cell or derived from a humancell. A cell can be a microbe cell or derived from a microbe cell. Acell can be a fungi cell or derived from a fungi cell. A cell can be aninsect cell. A cell can be an arthropod cell. A cell can be a protozoancell. A cell can be a helminth cell.

Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, aninduced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, asperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. afibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, aneuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell,etc.

Suitable cells include human embryonic stem cells, fetal cardiomyocytes,myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes,totipotent cells, pluripotent cells, blood stem cells, myoblasts, adultstem cells, bone marrow cells, mesenchymal cells, embryonic stem cells,parenchymal cells, epithelial cells, endothelial cells, mesothelialcells, fibroblasts, osteoblasts, chondrocytes, exogenous cells,endogenous cells, stem cells, hematopoietic stem cells, bone-marrowderived progenitor cells, myocardial cells, skeletal cells, fetal cells,undifferentiated cells, multi-potent progenitor cells, unipotentprogenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts,macrophages, capillary endothelial cells, xenogenic cells, allogeniccells, and post-natal stem cells.

In some cases, the cell is an immune cell, a neuron, an epithelial cell,and endothelial cell, or a stem cell. In some cases, the immune cell isa T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell,or a macrophage. In some cases, the immune cell is a cytotoxic T cell.In some cases, the immune cell is a helper T cell. In some cases, theimmune cell is a regulatory T cell (Treg).

In some cases, the cell is a stem cell. Stem cells include adult stemcells. Adult stem cells are also referred to as somatic stem cells.

Adult stem cells are resident in differentiated tissue, but retain theproperties of self-renewal and ability to give rise to multiple celltypes, usually cell types typical of the tissue in which the stem cellsare found. Numerous examples of somatic stem cells are known to those ofskill in the art, including muscle stem cells; hematopoietic stem cells;epithelial stem cells; neural stem cells; mesenchymal stem cells;mammary stem cells; intestinal stem cells; mesodermal stem cells;endothelial stem cells; olfactory stem cells; neural crest stem cells;and the like.

Stem cells of interest include mammalian stem cells, where the term“mammalian” refers to any animal classified as a mammal, includinghumans; non-human primates; domestic and farm animals; and zoo,laboratory, sports, or pet animals, such as dogs, horses, cats, cows,mice, rats, rabbits, etc. In some cases, the stem cell is a human stemcell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat)stem cell. In some cases, the stem cell is a non-human primate stemcell.

Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19,KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, andPPARGC1A.

In some embodiments, the stem cell is a hematopoietic stem cell (HSC).HSCs are mesoderm-derived cells that can be isolated from bone marrow,blood, cord blood, fetal liver and yolk sac. HSCs are characterized asCD34⁺ and CD3⁻. HSCs can repopulate the erythroid,neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic celllineages in vivo. In vitro, HSCs can be induced to undergo at least someself-renewing cell divisions and can be induced to differentiate to thesame lineages as is seen in vivo. As such, HSCs can be induced todifferentiate into one or more of erythroid cells, megakaryocytes,neutrophils, macrophages, and lymphoid cells.

In other embodiments, the stem cell is a neural stem cell (NSC). Neuralstem cells (NSCs) are capable of differentiating into neurons, and glia(including oligodendrocytes, and astrocytes). A neural stem cell is amultipotent stem cell which is capable of multiple divisions, and underspecific conditions can produce daughter cells which are neural stemcells, or neural progenitor cells that can be neuroblasts or glioblasts,e.g., cells committed to become one or more types of neurons and glialcells respectively. Methods of obtaining NSCs are known in the art.

In other embodiments, the stem cell is a mesenchymal stem cell (MSC).MSCs originally derived from the embryonal mesoderm and isolated fromadult bone marrow, can differentiate to form muscle, bone, cartilage,fat, marrow stroma, and tendon. Methods of isolating MSC are known inthe art; and any known method can be used to obtain MSC. See, e.g., U.S.Pat. No. 5,736,396, which describes isolation of human MSC.

A cell is in some cases a plant cell. A plant cell can be a cell of amonocotyledon. A cell can be a cell of a dicotyledon.

In some cases, the cell is a plant cell. For example, the cell can be acell of a major agricultural plant, e.g., Barley, Beans (Dry Edible),Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa),Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets,Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes,Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat(Spring), Wheat (Winter), and the like. As another example, the cell isa cell of a vegetable crops which include but are not limited to, e.g.,alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes,asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beettops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini),brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales),calabaza, cardoon, carrots, cauliflower, celery, chayote, chineseartichoke (crosnes), chinese cabbage, chinese celery, chinese chives,choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks,corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (peatips), donqua (winter melon), eggplant, endive, escarole, fiddle headferns, field cress, frisee, gai choy (chinese mustard), gailon, galanga(siam, thai ginger), garlic, ginger root, gobo, greens, hanover saladgreens, huauzontle, jerusalem artichokes, jicama, kale greens, kohlrabi,lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce(boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lollarossa), lettuce (oak leaf—green), lettuce (oak leaf—red), lettuce(processed), lettuce (red leaf), lettuce (romaine), lettuce (rubyromaine), lettuce (russian red mustard), linkok, lo bok, long beans,lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna,moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard,nagaimo, okra, ong choy, onions green, opo (long squash), ornamentalcorn, ornamental gourds, parsley, parsnips, peas, peppers (bell type),peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens,rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (seabean), sinqua (angled/ridged luffa), spinach, squash, straw bales,sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taroshoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes,tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric,turnip tops greens, turnips, water chestnuts, yampi, yams (names), yuchoy, yuca (cassava), and the like.

In some cases, the plant cell is a cell of a plant component such as aleaf, a stem, a root, a seed, a flower, pollen, an anther, an ovule, apedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod, anembryo, endosperm, an explant, a callus, or a shoot.

A cell is in some cases an arthropod cell. For example, the cell can bea cell of a sub-order, a family, a sub-family, a group, a sub-group, ora species of, e.g., chelicerata, Myriapodia, Hexipodia, Arachnida,insecta, Archaeognatha, thysanura, Palaeoptera, Ephemeroptera, odonata,anisoptera, zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera,orthoptera, Zoraptera, dermaptera, Dictyoptera, Notoptera,Grylloblattidae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera,Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera,hemiptera, Endopterygota or Holometabola, hymenoptera, coleoptera,Strepsiptera, Raphidioptera, megaloptera, neuroptera, Mecoptera,Siphonaptera, Diptera, Trichoptera, or lepidoptera.

A cell is in some cases an insect cell. For example, in some cases, thecell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea,a bee, a wasp, an ant, a louse, a moth, or a beetle.

Kits

The present disclosure provides a kit comprising a Cas12J system of thepresent disclosure, or a component of a Cas12J system of the presentdisclosure.

A kit of the present disclosure can comprise: a) a Cas12J polypeptide ofthe present disclosure and a Cas12J guide RNA; b) a Cas12J polypeptideof the present disclosure, a Cas12J guide RNA, and a donor templatenucleic acid; c) a Cas12J fusion polypeptide of the present disclosureand a Cas12J guide RNA; d) a Cas12J fusion polypeptide of the presentdisclosure, a Cas12J guide RNA, and a donor template nucleic acid; e) anmRNA encoding a Cas12J polypeptide of the present disclosure; and aCas12J guide RNA; f) an mRNA encoding a Cas12J polypeptide of thepresent disclosure, a Cas12J guide RNA, and a donor template nucleicacid; g) an mRNA encoding a Cas12J fusion polypeptide of the presentdisclosure; and a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusionpolypeptide of the present disclosure, a Cas12J guide RNA, and a donortemplate nucleic acid; i) a recombinant expression vector comprising anucleotide sequence encoding a Cas12J polypeptide of the presentdisclosure and a nucleotide sequence encoding a Cas12J guide RNA; j) arecombinant expression vector comprising a nucleotide sequence encodinga Cas12J polypeptide of the present disclosure, a nucleotide sequenceencoding a Cas12J guide RNA, and a nucleotide sequence encoding a donortemplate nucleic acid; k) a recombinant expression vector comprising anucleotide sequence encoding a Cas12J fusion polypeptide of the presentdisclosure and a nucleotide sequence encoding a Cas12J guide RNA; 1) arecombinant expression vector comprising a nucleotide sequence encodinga Cas12J fusion polypeptide of the present disclosure, a nucleotidesequence encoding a Cas12J guide RNA, and a nucleotide sequence encodinga donor template nucleic acid; m) a first recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure, and a second recombinant expression vectorcomprising a nucleotide sequence encoding a Cas12J guide RNA; n) a firstrecombinant expression vector comprising a nucleotide sequence encodinga Cas12J polypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jguide RNA; and a donor template nucleic acid; o) a first recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jfusion polypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jguide RNA; p) a first recombinant expression vector comprising anucleotide sequence encoding a Cas12J fusion polypeptide of the presentdisclosure, and a second recombinant expression vector comprising anucleotide sequence encoding a Cas12J guide RNA; and a donor templatenucleic acid; q) a recombinant expression vector comprising a nucleotidesequence encoding a Cas12J polypeptide of the present disclosure, anucleotide sequence encoding a first Cas12J guide RNA, and a nucleotidesequence encoding a second Cas12J guide RNA; or r) a recombinantexpression vector comprising a nucleotide sequence encoding a Cas12Jfusion polypeptide of the present disclosure, a nucleotide sequenceencoding a first Cas12J guide RNA, and a nucleotide sequence encoding asecond Cas12J guide RNA; or some variation of one of (a) through (r).

A kit of the present disclosure can comprise: a) a component, asdescribed above, of a Cas12J system of the present disclosure, or cancomprise a Cas12J system of the present disclosure; and b) one or moreadditional reagents, e.g., i) a buffer; ii) a protease inhibitor; iii) anuclease inhibitor; iv) a reagent required to develop or visualize adetectable label; v) a positive and/or negative control target DNA; vi)a positive and/or negative control Cas12J guide RNA; and the like. A kitof the present disclosure can comprise: a) a component, as describedabove, of a Cas12J system of the present disclosure, or can comprise aCas12J system of the present disclosure; and b) a therapeutic agent.

A kit of the present disclosure can comprise a recombinant expressionvector comprising: a) an insertion site for inserting a nucleic acidcomprising a nucleotide sequence encoding a portion of a Cas12J guideRNA that hybridizes to a target nucleotide sequence in a target nucleicacid; and b) a nucleotide sequence encoding the Cas12J-binding portionof a Cas12J guide RNA. A kit of the present disclosure can comprise arecombinant expression vector comprising: a) an insertion site forinserting a nucleic acid comprising a nucleotide sequence encoding aportion of a Cas12J guide RNA that hybridizes to a target nucleotidesequence in a target nucleic acid; b) a nucleotide sequence encoding theCas12J-binding portion of a Cas12J guide RNA; and c) a nucleotidesequence encoding a Cas12J polypeptide of the present disclosure.

Utility

A Cas12J polypeptide of the present disclosure, or a Cas12J fusionpolypeptide of the present disclosure, finds use in a variety of methods(e.g., in combination with a Cas12J guide RNA and in some cases furtherin combination with a donor template). For example, a Cas12J polypeptideof the present disclosure can be used to (i) modify (e.g., cleave, e.g.,nick; methylate; etc.) target nucleic acid (DNA or RNA; single strandedor double stranded); (ii) modulate transcription of a target nucleicacid; (iii) label a target nucleic acid; (iv) bind a target nucleic acid(e.g., for purposes of isolation, labeling, imaging, tracking, etc.);(v) modify a polypeptide (e.g., a histone) associated with a targetnucleic acid; and the like. Thus, the present disclosure provides amethod of modifying a target nucleic acid. In some cases, a method ofthe present disclosure for modifying a target nucleic acid comprisescontacting the target nucleic acid with: a) a Cas12J polypeptide of thepresent disclosure; and b) one or more (e.g., two) Cas12J guide RNAs. Insome cases, a method of the present disclosure for modifying a targetnucleic acid comprises contacting the target nucleic acid with: a) aCas12J polypeptide of the present disclosure; b) a Cas12J guide RNA; andc) a donor nucleic acid (e.g, a donor template). In some cases, thecontacting step is carried out in a cell in vitro. In some cases, thecontacting step is carried out in a cell in vivo. In some cases, thecontacting step is carried out in a cell ex vivo.

Because a method that uses a Cas12J polypeptide includes binding of theCas12J polypeptide to a particular region in a target nucleic acid (byvirtue of being targeted there by an associated Cas12J guide RNA), themethods are generally referred to herein as methods of binding (e.g., amethod of binding a target nucleic acid). However, it is to beunderstood that in some cases, while a method of binding may result innothing more than binding of the target nucleic acid, in other cases,the method can have different final results (e.g., the method can resultin modification of the target nucleic acid, e.g.,cleavage/methylation/etc., modulation of transcription from the targetnucleic acid; modulation of translation of the target nucleic acid;genome editing; modulation of a protein associated with the targetnucleic acid; isolation of the target nucleic acid; etc.).

For examples of suitable methods, see, for example, Jinek et al.,Science. 2012 Aug. 17; 337(6096):816-21; Chylinski et al., RNA Biol.2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805;Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Jineket al., Elife. 2013; 2:e00471; Pattanayak et al., Nat Biotechnol. 2013September; 31(9):839-43; Qi et al, Cell. 2013 Feb. 28; 152(5):1173-83;Wang et al., Cell. 2013 May 9; 153(4):910-8; Auer et al., Genome Res.2013 Oct. 31; Chen et al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e19;Cheng et al., Cell Res. 2013 October; 23(10):1163-71; Cho et al.,Genetics. 2013 November; 195(3):1177-80; DiCarlo et al., Nucleic AcidsRes. 2013 April; 41(7):4336-43; Dickinson et al., Nat Methods. 2013October; 10(10):1028-34; Ebina et al., Sci Rep. 2013; 3:2510; Fujii etal, Nucleic Acids Res. 2013 Nov. 1; 41(20):e187; Hu et al., Cell Res.2013 November; 23(11):1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov.1; 41(20):e188; Larson et al., Nat Protoc. 2013 November; 8(11):2180-96;Mali et. at., Nat Methods. 2013 October; 10(10):957-63; Nakayama et al.,Genesis. 2013 December; 51(12):835-43; Ran et al., Nat Protoc. 2013November; 8(11):2281-308; Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9;Upadhyay et al., G3 (Bethesda). 2013 Dec. 9; 3(12):2233-8; Walsh et al.,Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15514-5; Xie et al., MolPlant. 2013 Oct. 9; Yang et al., Cell. 2013 Sep. 12; 154(6):1370-9; andU.S. patents and patent applications: U.S. Pat. Nos. 8,906,616;8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965;8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006;20140179770; 20140186843; 20140186919; 20140186958; 20140189896;20140227787; 20140234972; 20140242664; 20140242699; 20140242700;20140242702; 20140248702; 20140256046; 20140273037; 20140273226;20140273230; 20140273231; 20140273232; 20140273233; 20140273234;20140273235; 20140287938; 20140295556; 20140295557; 20140298547;20140304853; 20140309487; 20140310828; 20140310830; 20140315985;20140335063; 20140335620; 20140342456; 20140342457; 20140342458;20140349400; 20140349405; 20140356867; 20140356956; 20140356958;20140356959; 20140357523; 20140357530; 20140364333; and 20140377868;each of which is hereby incorporated by reference in its entirety.

For example, the present disclosure provides (but is not limited to)methods of cleaving a target nucleic acid; methods of editing a targetnucleic acid; methods of modulating transcription from a target nucleicacid; methods of isolating a target nucleic acid, methods of binding atarget nucleic acid, methods of imaging a target nucleic acid, methodsof modifying a target nucleic acid, and the like.

As used herein, the terms/phrases “contact a target nucleic acid” and“contacting a target nucleic acid”, for example, with a Cas12Jpolypeptide or with a Cas12J fusion polypeptide, etc., encompass allmethods for contacting the target nucleic acid. For example, a Cas12Jpolypeptide can be provided to a cell as protein, RNA (encoding theCas12J polypeptide), or DNA (encoding the Cas12J polypeptide); while aCas12J guide RNA can be provided as a guide RNA or as a nucleic acidencoding the guide RNA. As such, when, for example, performing a methodin a cell (e.g., inside of a cell in vitro, inside of a cell in vivo,inside of a cell ex vivo), a method that includes contacting the targetnucleic acid encompasses the introduction into the cell of any or all ofthe components in their active/final state (e.g., in the form of aprotein(s) for Cas12J polypeptide; in the form of a protein for a Cas12Jfusion polypeptide; in the form of an RNA in some cases for the guideRNA), and also encompasses the introduction into the cell of one or morenucleic acids encoding one or more of the components (e.g., nucleicacid(s) comprising nucleotide sequence(s) encoding a Cas12J polypeptideor a Cas12J fusion polypeptide, nucleic acid(s) comprising nucleotidesequence(s) encoding guide RNA(s), nucleic acid comprising a nucleotidesequence encoding a donor template, and the like). Because the methodscan also be performed in vitro outside of a cell, a method that includescontacting a target nucleic acid, (unless otherwise specified)encompasses contacting outside of a cell in vitro, inside of a cell invitro, inside of a cell in vivo, inside of a cell ex vivo, etc.

In some cases, a method of the present disclosure for modifying a targetnucleic acid comprises introducing into a target cell a Cas12J locus,e.g., a nucleic acid comprising a nucleotide sequence encoding a Cas12Jpolypeptide as well as nucleotide sequences of about 1 kilobase (kb) to5 kb in length surrounding the Cas12J-encoding nucleotide sequence froma cell (e.g., in some cases a cell that in its natural state (the statein which it occurs in nature) comprises a Cas12J locus) comprising aCas12J locus, where the target cell does not normally (in its naturalstate) comprise a Cas12J locus. However, one or more spacer sequences,encoding guide sequences for the encoded crRNA(s), can be modified suchthat one or more target sequences of interest are targeted. Thus, forexample, in some cases, a method of the present disclosure for modifyinga target nucleic acid comprises introducing into a target cell a Cas12Jlocus, e.g., a nucleic acid obtained from a source cell (e.g., in somecases a cell that in its natural state (the state in which it occurs innature) comprises a Cas12J locus), where the nucleic acid has a lengthof from 100 nucleotides (nt) to 5 kb in length (e.g., from 100 nt to 500nt, from 500 nt to 1 kb, from 1 kb to 1.5 kb, from 1.5 kb to 2 kb, from2 kb to 2.5 kb, from 2.5 kb to 3 kb, from 3 kb to 3.5 kb, from 3.5 kb to4 kb, or from 4 kb to 5 kb in length) and comprises a nucleotidesequence encoding a Cas12J polypeptide. As noted above, in some suchcases, one or more spacer sequences, encoding guide sequences for theencoded crRNA(s), can be modified such that one or more target sequencesof interest are targeted. In some cases, the method comprisesintroducing into a target cell: i) a Cas12J locus; and ii) a donor DNAtemplate. In some cases, the target nucleic acid is in a cell-freecomposition in vitro. In some cases, the target nucleic acid is presentin a target cell. In some cases, the target nucleic acid is present in atarget cell, where the target cell is a prokaryotic cell. In some cases,the target nucleic acid is present in a target cell, where the targetcell is a eukaryotic cell. In some cases, the target nucleic acid ispresent in a target cell, where the target cell is a mammalian cell. Insome cases, the target nucleic acid is present in a target cell, wherethe target cell is a plant cell.

In some cases, a method of the present disclosure for modifying a targetnucleic acid comprises contacting a target nucleic acid with a Cas12Jpolypeptide of the present disclosure, or with a Cas12J fusionpolypeptide of the present disclosure. In some cases, a method of thepresent disclosure for modifying a target nucleic acid comprisescontacting a target nucleic acid with a Cas12J polypeptide and a Cas12Jguide RNA. In some cases, a method of the present disclosure formodifying a target nucleic acid comprises contacting a target nucleicacid with a Cas12J polypeptide, a first Cas12J guide RNA, and a secondCas12J guide RNA In some cases, a method of the present disclosure formodifying a target nucleic acid comprises contacting a target nucleicacid with a Cas12J polypeptide of the present disclosure and a Cas12Jguide RNA and a donor DNA template.

Target Nucleic Acids and Target Cells of Interest

A Cas12J polypeptide of the present disclosure, or a Cas12J fusionpolypeptide of the present disclosure, when bound to a Cas12J guide RNA,can bind to a target nucleic acid, and in some cases, can bind to andmodify a target nucleic acid. A target nucleic acid can be any nucleicacid (e.g., DNA, RNA), can be double stranded or single stranded, can beany type of nucleic acid (e.g., a chromosome (genomic DNA), derived froma chromosome, chromosomal DNA, plasmid, viral, extracellular,intracellular, mitochondrial, chloroplast, linear, circular, etc.) andcan be from any organism (e.g., as long as the Cas12J guide RNAcomprises a nucleotide sequence that hybridizes to a target sequence ina target nucleic acid, such that the target nucleic acid can betargeted).

A target nucleic acid can be DNA or RNA. A target nucleic acid can bedouble stranded (e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA,ssDNA). In some cases, a target nucleic acid is single stranded. In somecases, a target nucleic acid is a single stranded RNA (ssRNA). In somecases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.)is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), longnon-coding RNA (lncRNA), and microRNA (miRNA). In some cases, a targetnucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). Asnoted above, in some cases, a target nucleic acid is single stranded.

A target nucleic acid can be located anywhere, for example, outside of acell in vitro, inside of a cell in vitro, inside of a cell in vivo,inside of a cell ex vivo. Suitable target cells (which can comprisetarget nucleic acids such as genomic DNA) include, but are not limitedto: a bacterial cell; an archaeal cell; a cell of a single-celleukaryotic organism; a plant cell; an algal cell, e.g., Botryococcusbraunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorellapyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell(e.g., a yeast cell); an animal cell; a cell from an invertebrate animal(e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cellof an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); acell of an arachnid (e.g., a spider; a tick; etc.); a cell from avertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, amammal); a cell from a mammal (e.g., a cell from a rodent; a cell from ahuman; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse,a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate(e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat,etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephantseal, a dolphin, a sea lion; etc.) and the like. Any type of cell may beof interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, aninduced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, asperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somaticcell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell,a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivoembryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell,4-cell, 8-cell, etc. stage zebrafish embryo; etc.).

Cells may be from established cell lines or they may be primary cells,where “primary cells”, “primary cell lines”, and “primary cultures” areused interchangeably herein to refer to cells and cells cultures thathave been derived from a subject and allowed to grow in vitro for alimited number of passages, i.e. splittings, of the culture. Forexample, primary cultures are cultures that may have been passaged 0times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but notenough times go through the crisis stage. Typically, the primary celllines are maintained for fewer than 10 passages in vitro. Target cellscan be unicellular organisms and/or can be grown in culture. If thecells are primary cells, they may be harvest from an individual by anyconvenient method. For example, leukocytes may be conveniently harvestedby apheresis, leukocytapheresis, density gradient separation, etc.,while cells from tissues such as skin, muscle, bone marrow, spleen,liver, pancreas, lung, intestine, stomach, etc. can be convenientlyharvested by biopsy.

In some of the above applications, the subject methods may be employedto induce target nucleic acid cleavage, target nucleic acidmodification, and/or to bind target nucleic acids (e.g., forvisualization, for collecting and/or analyzing, etc.) in mitotic orpost-mitotic cells in vivo and/or ex vivo and/or in vitro (e.g., todisrupt production of a protein encoded by a targeted mRNA, to cleave orotherwise modify target DNA, to genetically modify a target cell, andthe like). Because the guide RNA provides specificity by hybridizing totarget nucleic acid, a mitotic and/or post-mitotic cell of interest inthe disclosed methods may include a cell from any organism (e.g. abacterial cell, an archaeal cell, a cell of a single-cell eukaryoticorganism, a plant cell, an algal cell, e.g., Botryococcus braunii,Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorellapyrenoidosa, Sargassum patens, C. agardh, and the like, a fungal cell(e.g., a yeast cell), an animal cell, a cell from an invertebrate animal(e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from avertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cellfrom a mammal, a cell from a rodent, a cell from a human, etc.). In somecases, a subject Cas12J protein (and/or nucleic acid encoding theprotein such as DNA and/or RNA), and/or Cas12J guide RNA (and/or a DNAencoding the guide RNA), and/or donor template, and/or RNP can beintroduced into an individual (i.e., the target cell can be in vivo)(e.g., a mammal, a rat, a mouse, a pig, a primate, a non-human primate,a human, etc.). In some case, such an administration can be for thepurpose of treating and/or preventing a disease, e.g., by editing thegenome of targeted cells.

Plant cells include cells of a monocotyledon, and cells of adicotyledon. The cells can be root cells, leaf cells, cells of thexylem, cells of the phloem, cells of the cambium, apical meristem cells,parenchyma cells, collenchyma cells, sclerenchyma cells, and the like.Plant cells include cells of agricultural crops such as wheat, corn,rice, sorghum, millet, soybean, etc. Plant cells include cells ofagricultural fruit and nut plants, e.g., plant that produce apricots,oranges, lemons, apples, plums, pears, almonds, etc.

Additional examples of target cells are listed above in the sectiontitled “Modified cells.” Non-limiting examples of cells (target cells)include: a prokaryotic cell, eukaryotic cell, a bacterial cell, anarchaeal cell, a cell of a single-cell eukaryotic organism, a protozoacell, a cell from a plant (e.g., cells from plant crops, fruits,vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice,cassava, sugarcane, pumpkin, hay, potatos, cotton, cannabis, tobacco,flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses,hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), analgal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii,Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeastcell, a cell from a mushroom), an animal cell, a cell from aninvertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode,etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile,bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, acow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-humanprimate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.),and the like. In some cases, the cell is a cell that does not originatefrom a natural organism (e.g., the cell can be a synthetically madecell; also referred to as an artificial cell).

A cell can be an in vitro cell (e.g., established cultured cell line). Acell can be an ex vivo cell (cultured cell from an individual). A cellcan be and in vivo cell (e.g., a cell in an individual). A cell can bean isolated cell. A cell can be a cell inside of an organism. A cell canbe an organism. A cell can be a cell in a cell culture (e.g., in vitrocell culture). A cell can be one of a collection of cells. A cell can bea prokaryotic cell or derived from a prokaryotic cell. A cell can be abacterial cell or can be derived from a bacterial cell. A cell can be anarchaeal cell or derived from an archaeal cell. A cell can be aeukaryotic cell or derived from a eukaryotic cell. A cell can be a plantcell or derived from a plant cell. A cell can be an animal cell orderived from an animal cell. A cell can be an invertebrate cell orderived from an invertebrate cell. A cell can be a vertebrate cell orderived from a vertebrate cell. A cell can be a mammalian cell orderived from a mammalian cell. A cell can be a rodent cell or derivedfrom a rodent cell. A cell can be a human cell or derived from a humancell. A cell can be a microbe cell or derived from a microbe cell. Acell can be a fungi cell or derived from a fungi cell. A cell can be aninsect cell. A cell can be an arthropod cell. A cell can be a protozoancell. A cell can be a helminth cell.

Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, aninduced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, asperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. afibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, aneuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell,etc.

Suitable cells include human embryonic stem cells, fetal cardiomyocytes,myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes,totipotent cells, pluripotent cells, blood stem cells, myoblasts, adultstem cells, bone marrow cells, mesenchymal cells, embryonic stem cells,parenchymal cells, epithelial cells, endothelial cells, mesothelialcells, fibroblasts, osteoblasts, chondrocytes, exogenous cells,endogenous cells, stem cells, hematopoietic stem cells, bone-marrowderived progenitor cells, myocardial cells, skeletal cells, fetal cells,undifferentiated cells, multi-potent progenitor cells, unipotentprogenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts,macrophages, capillary endothelial cells, xenogenic cells, allogeniccells, and post-natal stem cells.

In some cases, the cell is an immune cell, a neuron, an epithelial cell,and endothelial cell, or a stem cell. In some cases, the immune cell isa T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell,or a macrophage. In some cases, the immune cell is a cytotoxic T cell.In some cases, the immune cell is a helper T cell. In some cases, theimmune cell is a regulatory T cell (Treg).

In some cases, the cell is a stem cell. Stem cells include adult stemcells. Adult stem cells are also referred to as somatic stem cells.

Adult stem cells are resident in differentiated tissue, but retain theproperties of self-renewal and ability to give rise to multiple celltypes, usually cell types typical of the tissue in which the stem cellsare found. Numerous examples of somatic stem cells are known to those ofskill in the art, including muscle stem cells; hematopoietic stem cells;epithelial stem cells; neural stem cells; mesenchymal stem cells;mammary stem cells; intestinal stem cells; mesodermal stem cells;endothelial stem cells; olfactory stem cells; neural crest stem cells;and the like.

Stem cells of interest include mammalian stem cells, where the term“mammalian” refers to any animal classified as a mammal, includinghumans; non-human primates; domestic and farm animals; and zoo,laboratory, sports, or pet animals, such as dogs, horses, cats, cows,mice, rats, rabbits, etc. In some cases, the stem cell is a human stemcell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat)stem cell. In some cases, the stem cell is a non-human primate stemcell.

Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19,KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, andPPARGC1A.

In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCsare mesoderm-derived cells that can be isolated from bone marrow, blood,cord blood, fetal liver and yolk sac. HSCs are characterized as CD34⁺and CD3⁻. HSCs can repopulate the erythroid, neutrophil-macrophage,megakaryocyte and lymphoid hematopoietic cell lineages in vivo. Invitro, HSCs can be induced to undergo at least some self-renewing celldivisions and can be induced to differentiate to the same lineages as isseen in vivo. As such, HSCs can be induced to differentiate into one ormore of erythroid cells, megakaryocytes, neutrophils, macrophages, andlymphoid cells.

In other embodiments, the stem cell is a neural stem cell (NSC). Neuralstem cells (NSCs) are capable of differentiating into neurons, and glia(including oligodendrocytes, and astrocytes). A neural stem cell is amultipotent stem cell which is capable of multiple divisions, and underspecific conditions can produce daughter cells which are neural stemcells, or neural progenitor cells that can be neuroblasts or glioblasts,e.g., cells committed to become one or more types of neurons and glialcells respectively. Methods of obtaining NSCs are known in the art.

In other embodiments, the stem cell is a mesenchymal stem cell (MSC).MSCs originally derived from the embryonal mesoderm and isolated fromadult bone marrow, can differentiate to form muscle, bone, cartilage,fat, marrow stroma, and tendon. Methods of isolating MSC are known inthe art; and any known method can be used to obtain MSC. See, e.g., U.S.Pat. No. 5,736,396, which describes isolation of human MSC.

A cell is in some cases a plant cell. A plant cell can be a cell of amonocotyledon. A cell can be a cell of a dicotyledon.

In some cases, the cell is a plant cell. For example, the cell can be acell of a major agricultural plant, e.g., Barley, Beans (Dry Edible),Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa),Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets,Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes,Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat(Spring), Wheat (Winter), and the like. As another example, the cell isa cell of a vegetable crops which include but are not limited to, e.g.,alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes,asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beettops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini),brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales),calabaza, cardoon, carrots, cauliflower, celery, chayote, chineseartichoke (crosnes), chinese cabbage, chinese celery, chinese chives,choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks,corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (peatips), donqua (winter melon), eggplant, endive, escarole, fiddle headferns, field cress, frisee, gai choy (chinese mustard), gailon, galanga(siam, thai ginger), garlic, ginger root, gobo, greens, hanover saladgreens, huauzontle, jerusalem artichokes, jicama, kale greens, kohlrabi,lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce(boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lollarossa), lettuce (oak leaf—green), lettuce (oak leaf—red), lettuce(processed), lettuce (red leaf), lettuce (romaine), lettuce (rubyromaine), lettuce (russian red mustard), linkok, lo bok, long beans,lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna,moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard,nagaimo, okra, ong choy, onions green, opo (long squash), ornamentalcorn, ornamental gourds, parsley, parsnips, peas, peppers (bell type),peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens,rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (seabean), sinqua (angled/ridged luffa), spinach, squash, straw bales,sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taroshoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes,tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric,turnip tops greens, turnips, water chestnuts, yampi, yams, yu choy, yuca(cassava), and the like.

A cell is in some cases an arthropod cell. For example, the cell can bea cell of a sub-order, a family, a sub-family, a group, a sub-group, ora species of, e.g., chelicerata, Myriapodia, Hexipodia, Arachnida,insecta, Archaeognatha, thysanura, Palaeoptera, Ephemeroptera, odonata,anisoptera, zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera,orthoptera, Zoraptera, dermaptera, Dictyoptera, Notoptera,Grylloblattidae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera,Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera,hemiptera, Endopterygota or Holometabola, hymenoptera, coleoptera,Strepsiptera, Raphidioptera, megaloptera, neuroptera, Mecoptera,Siphonaptera, Diptera, Trichoptera, or lepidoptera.

A cell is in some cases an insect cell. For example, in some cases, thecell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea,a bee, a wasp, an ant, a louse, a moth, or a beetle.

Introducing Components into a Target Cell

A Cas12J guide RNA (or a nucleic acid comprising a nucleotide sequenceencoding same), and/or a Cas12J fusion polypeptide (or a nucleic acidcomprising a nucleotide sequence encoding same) and/or a donorpolynucleotide can be introduced into a host cell by any of a variety ofwell-known methods.

Methods of introducing a nucleic acid into a cell are known in the art,and any convenient method can be used to introduce a nucleic acid (e.g.,an expression construct) into a target cell (e.g., eukaryotic cell,human cell, stem cell, progenitor cell, and the like). Suitable methodsare described in more detail elsewhere herein and include e.g., viral orbacteriophage infection, transfection, conjugation, protoplast fusion,lipofection, electroporation, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro injection,nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., alAdv Drug Deliv Rev. 2012 Sep. 13. pii: 50169-409X(12)00283-9. doi:10.1016/j.addr.2012.09.023), and the like. Any or all of the componentscan be introduced into a cell as a composition (e.g., including anyconvenient combination of: a Cas12J polypeptide, a Cas12J guide RNA, adonor polynucleotide, etc.) using known methods, e.g., such asnucleofection.

Donor Polynucleotide (Donor Template)

Guided by a Cas12J guide RNA, a Cas12J protein in some cases generatessite-specific double strand breaks (DSBs) or single strand breaks (SSBs)(e.g., when the Cas12J protein is a nickase variant) withindouble-stranded DNA (dsDNA) target nucleic acids, which are repairedeither by non-homologous end joining (NHEJ) or homology-directedrecombination (HDR).

In some cases, contacting a target DNA (with a Cas12J protein and aCas12J guide RNA) occurs under conditions that are permissive fornonhomologous end joining or homology-directed repair. Thus, in somecases, a subject method includes contacting the target DNA with a donorpolynucleotide (e.g., by introducing the donor polynucleotide into acell), wherein the donor polynucleotide, a portion of the donorpolynucleotide, a copy of the donor polynucleotide, or a portion of acopy of the donor polynucleotide integrates into the target DNA. In somecases, the method does not comprise contacting a cell with a donorpolynucleotide, and the target DNA is modified such that nucleotideswithin the target DNA are deleted.

In some cases, Cas12J guide RNA (or DNA encoding same) and a Cas12Jprotein (or a nucleic acid encoding same, such as an RNA or a DNA, e.g,one or more expression vectors) are coadministered (e.g., contacted witha target nucleic acid, administered to cells, etc.) with a donorpolynucleotide sequence that includes at least a segment with homologyto the target DNA sequence, the subject methods may be used to add, i.e.insert or replace, nucleic acid material to a target DNA sequence (e.g.to “knock in” a nucleic acid, e.g., one that encodes for a protein, ansiRNA, an miRNA, etc.), to add a tag (e.g., 6×His, a fluorescent protein(e.g., a green fluorescent protein; a yellow fluorescent protein, etc.),hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene(e.g. promoter, polyadenylation signal, internal ribosome entry sequence(IRES), 2A peptide, start codon, stop codon, splice signal, localizationsignal, etc.), to modify a nucleic acid sequence (e.g., introduce amutation, remove a disease causing mutation by introducing a correctsequence), and the like. As such, a complex comprising a Cas12J guideRNA and Cas12J protein is useful in any in vitro or in vivo applicationin which it is desirable to modify DNA in a site-specific, i.e.“targeted”, way, for example gene knock-out, gene knock-in, geneediting, gene tagging, etc., as used in, for example, gene therapy, e.g.to treat a disease or as an antiviral, antipathogenic, or anticancertherapeutic, the production of genetically modified organisms inagriculture, the large scale production of proteins by cells fortherapeutic, diagnostic, or research purposes, the induction of iPScells, biological research, the targeting of genes of pathogens fordeletion or replacement, etc.

In applications in which it is desirable to insert a polynucleotidesequence into he genome where a target sequence is cleaved, a donorpolynucleotide (a nucleic acid comprising a donor sequence) can also beprovided to the cell. By a “donor sequence” or “donor polynucleotide” or“donor template” it is meant a nucleic acid sequence to be inserted atthe site cleaved by the Cas12J protein (e.g., after dsDNA cleavage,after nicking a target DNA, after dual nicking a target DNA, and thelike). The donor polynucleotide can contain sufficient homology to agenomic sequence at the target site, e.g. 70%, 80%, 85%, 90%, 95%, or100% homology with the nucleotide sequences flanking the target site,e.g. within about 50 bases or less of the target site, e.g. within about30 bases, within about 15 bases, within about 10 bases, within about 5bases, or immediately flanking the target site, to supporthomology-directed repair between it and the genomic sequence to which itbears homology. Approximately 25, 50, 100, or 200 nucleotides, or morethan 200 nucleotides, of sequence homology between a donor and a genomicsequence (or any integral value between 10 and 200 nucleotides, or more)can support homology-directed repair. Donor polynucleotides can be ofany length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100nucleotides or more, 250 nucleotides or more, 500 nucleotides or more,1000 nucleotides or more, 5000 nucleotides or more, etc.

The donor sequence is typically not identical to the genomic sequencethat it replaces. Rather, the donor sequence may contain at least one ormore single base changes, insertions, deletions, inversions orrearrangements with respect to the genomic sequence, so long assufficient homology is present to support homology-directed repair(e.g., for gene correction, e.g., to convert a disease-causing base pairto a non disease-causing base pair). In some embodiments, the donorsequence comprises a non-homologous sequence flanked by two regions ofhomology, such that homology-directed repair between the target DNAregion and the two flanking sequences results in insertion of thenon-homologous sequence at the target region. Donor sequences may alsocomprise a vector backbone containing sequences that are not homologousto the DNA region of interest and that are not intended for insertioninto the DNA region of interest. Generally, the homologous region(s) ofa donor sequence will have at least 50% sequence identity to a genomicsequence with which recombination is desired. In certain embodiments,60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity ispresent. Any value between 1% and 100% sequence identity can be present,depending upon the length of the donor polynucleotide.

The donor sequence may comprise certain sequence differences as comparedto the genomic sequence, e.g. restriction sites, nucleotidepolymorphisms, selectable markers (e.g., drug resistance genes,fluorescent proteins, enzymes etc.), etc., which may be used to assessfor successful insertion of the donor sequence at the cleavage site orin some cases may be used for other purposes (e.g., to signifyexpression at the targeted genomic locus). In some cases, if located ina coding region, such nucleotide sequence differences will not changethe amino acid sequence, or will make silent amino acid changes (i.e.,changes which do not affect the structure or function of the protein).Alternatively, these sequences differences may include flankingrecombination sequences such as FLPs, loxP sequences, or the like, thatcan be activated at a later time for removal of the marker sequence.

In some cases, the donor sequence is provided to the cell assingle-stranded DNA. In some cases, the donor sequence is provided tothe cell as double-stranded DNA. It may be introduced into a cell inlinear or circular form. If introduced in linear form, the ends of thedonor sequence may be protected (e.g., from exonucleolytic degradation)by any convenient method and such methods are known to those of skill inthe art. For example, one or more dideoxynucleotide residues can beadded to the 3′ terminus of a linear molecule and/or self-complementaryoligonucleotides can be ligated to one or both ends. See, for example,Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al.(1996) Science 272:886-889. Additional methods for protecting exogenouspolynucleotides from degradation include, but are not limited to,addition of terminal amino group(s) and the use of modifiedinternucleotide linkages such as, for example, phosphorothioates,phosphoramidates, and O-methyl ribose or deoxyribose residues. As analternative to protecting the termini of a linear donor sequence,additional lengths of sequence may be included outside of the regions ofhomology that can be degraded without impacting recombination. A donorsequence can be introduced into a cell as part of a vector moleculehaving additional sequences such as, for example, replication origins,promoters and genes encoding antibiotic resistance. Moreover, donorsequences can be introduced as naked nucleic acid, as nucleic acidcomplexed with an agent such as a liposome or poloxamer, or can bedelivered by viruses (e.g., adenovirus, AAV), as described elsewhereherein for nucleic acids encoding a Cas12J guide RNA and/or a Cas12Jfusion polypeptide and/or donor polynucleotide.

Detection Methods

A Cas12J polypeptide of the present disclosure can promiscuously cleavenon-targeted single stranded DNA (ssDNA) once activated by detection ofa target DNA (double or single stranded). Once a Cas12J polypeptide ofthe present disclosure is activated by a guide RNA, which occurs whenthe guide RNA hybridizes to a target sequence of a target DNA (i.e., thesample includes the targeted DNA), the Cas12J polypeptide becomes anuclease that promiscuously cleaves ssDNAs (i.e., the nuclease cleavesnon-target ssDNAs, i.e., ssDNAs to which the guide sequence of the guideRNA does not hybridize). Thus, when the target DNA is present in thesample (e.g., in some cases above a threshold amount), the result iscleavage of ssDNAs in the sample, which can be detected using anyconvenient detection method (e.g., using a labeled single strandeddetector DNA). Cleavage of non-target nucleic acid is referred to as“trans cleavage.” In some cases, a Cas12J effector polypeptide of thepresent disclosure mediates trans cleavage of ssDNA, but not ssRNA.

Provided are compositions and methods for detecting a target DNA (doublestranded or single stranded) in a sample. In some cases, a detector DNAis used that is single stranded (ssDNA) and does not hybridize with theguide sequence of the guide RNA (i.e., the detector ssDNA is anon-target ssDNA). Such methods can include (a) contacting the samplewith: (i) a Cas12J polypeptide of the present disclosure; (ii) a guideRNA comprising: a region that binds to the Cas12J polypeptide, and aguide sequence that hybridizes with the target DNA; and (iii) a detectorDNA that is single stranded and does not hybridize with the guidesequence of the guide RNA; and (b) measuring a detectable signalproduced by cleavage of the single stranded detector DNA by the Cas12Jpolypeptide, thereby detecting the target DNA. As noted above, once aCas12J polypeptide of the present disclosure is activated by a guideRNA, which occurs when the sample includes a target DNA to which theguide RNA hybridizes (i.e., the sample includes the targeted targetDNA), the Cas12J polypeptide is activated and functions as anendoribonuclease that non-specifically cleaves ssDNAs (includingnon-target ssDNAs) present in the sample. Thus, when the targeted targetDNA is present in the sample (e.g., in some cases above a thresholdamount), the result is cleavage of ssDNA (including non-target ssDNA) inthe sample, which can be detected using any convenient detection method(e.g., using a labeled detector ssDNA).

Also provided are compositions and methods for cleaving single strandedDNAs (ssDNAs) (e.g., non-target ssDNAs). Such methods can includecontacting a population of nucleic acids, wherein said populationcomprises a target DNA and a plurality of non-target ssDNAs, with: (i) aCas12J polypeptide of the present disclosure; and (ii) a guide RNAcomprising: a region that binds to the Cas12J polypeptide and a guidesequence that hybridizes with the target DNA, wherein the Cas12Jpolypeptide cleaves non-target ssDNAs of said plurality. Such a methodcan be used, e.g., to cleave foreign ssDNAs (e.g., viral DNAs) in acell.

The contacting step of a subject method can be carried out in acomposition comprising divalent metal ions. The contacting step can becarried out in an acellular environment, e.g., outside of a cell. Thecontacting step can be carried out inside a cell. The contacting stepcan be carried out in a cell in vitro. The contacting step can becarried out in a cell ex vivo. The contacting step can be carried out ina cell in vivo.

The guide RNA can be provided as RNA or as a nucleic acid encoding theguide RNA (e.g., a DNA such as a recombinant expression vector). TheCas12J polypeptide can be provided as a protein or as a nucleic acidencoding the protein (e.g., an mRNA, a DNA such as a recombinantexpression vector). In some cases, two or more (e.g., 3 or more, 4 ormore, 5 or more, or 6 or more) guide RNAs can be provided by (e.g.,using a precursor guide RNA array, which can be cleaved by the Cas12Jeffector protein into individual (“mature”) guide RNAs).

In some cases (e.g., when contacting with a guide RNA and a Cas12Jpolypeptide of the present disclosure, the sample is contacted for 2hours or less (e.g., 1.5 hours or less, 1 hour or less, 40 minutes orless, 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5minutes or less, or 1 minute or less) prior to the measuring step. Forexample, in some cases the sample is contacted for 40 minutes or lessprior to the measuring step. In some cases, the sample is contacted for20 minutes or less prior to the measuring step. In some cases, thesample is contacted for 10 minutes or less prior to the measuring step.In some cases, the sample is contacted for 5 minutes or less prior tothe measuring step. In some cases, the sample is contacted for 1 minuteor less prior to the measuring step. In some cases, the sample iscontacted for from 50 seconds to 60 seconds prior to the measuring step.In some cases, the sample is contacted for from 40 seconds to 50 secondsprior to the measuring step. In some cases, the sample is contacted forfrom 30 seconds to 40 seconds prior to the measuring step. In somecases, the sample is contacted for from 20 seconds to 30 seconds priorto the measuring step. In some cases, the sample is contacted for from10 seconds to 20 seconds prior to the measuring step.

A method of the present disclosure for detecting a target DNA(single-stranded or double-stranded) in a sample can detect a target DNAwith a high degree of sensitivity. In some cases, a method of thepresent disclosure can be used to detect a target DNA present in asample comprising a plurality of DNAs (including the target DNA and aplurality of non-target DNAs), where the target DNA is present at one ormore copies per 10⁷ non-target DNAs (e.g., one or more copies per 10⁶non-target DNAs, one or more copies per 10⁵ non-target DNAs, one or morecopies per 10⁴ non-target DNAs, one or more copies per 10³ non-targetDNAs, one or more copies per 10² non-target DNAs, one or more copies per50 non-target DNAs, one or more copies per 20 non-target DNAs, one ormore copies per 10 non-target DNAs, or one or more copies per 5non-target DNAs). In some cases, a method of the present disclosure canbe used to detect a target DNA present in a sample comprising aplurality of DNAs (including the target DNA and a plurality ofnon-target DNAs), where the target DNA is present at one or more copiesper 10¹⁸ non-target DNAs (e.g., one or more copies per 10¹⁵ non-targetDNAs, one or more copies per 10¹² non-target DNAs, one or more copiesper 10⁹ non-target DNAs, one or more copies per 10⁶ non-target DNAs, oneor more copies per 10⁵ non-target DNAs, one or more copies per 10⁴non-target DNAs, one or more copies per 10³ non-target DNAs, one or morecopies per 10² non-target DNAs, one or more copies per 50 non-targetDNAs, one or more copies per 20 non-target DNAs, one or more copies per10 non-target DNAs, or one or more copies per 5 non-target DNAs).

In some cases, a method of the present disclosure can detect a targetDNA present in a sample, where the target DNA is present at from onecopy per 10⁷ non-target DNAs to one copy per 10 non-target DNAs (e.g.,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁶ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10 non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10 non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10³ non-target DNAs,or from 1 copy per 10⁵ non-target DNAs to 1 copy per 10⁴ non-targetDNAs).

In some cases, a method of the present disclosure can detect a targetDNA present in a sample, where the target DNA is present at from onecopy per 10¹⁸ non-target DNAs to one copy per 10 non-target DNAs (e.g.,from 1 copy per 10¹⁸ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10¹⁵ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10¹² non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁹ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁶ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10 non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10 non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10³ non-target DNAs,or from 1 copy per 10⁵ non-target DNAs to 1 copy per 10⁴ non-targetDNAs).

In some cases, a method of the present disclosure can detect a targetDNA present in a sample, where the target DNA is present at from onecopy per 10⁷ non-target DNAs to one copy per 100 non-target DNAs (e.g.,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁷ non-target DNAs to 1 copy per 10⁶ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 100 non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10³ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁴ non-target DNAs,from 1 copy per 10⁶ non-target DNAs to 1 copy per 10⁵ non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 100 non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10² non-target DNAs,from 1 copy per 10⁵ non-target DNAs to 1 copy per 10³ non-target DNAs,or from 1 copy per 10⁵ non-target DNAs to 1 copy per 10⁴ non-targetDNAs).

In some cases, the threshold of detection, for a subject method ofdetecting a target DNA in a sample, is 10 nM or less. The term“threshold of detection” is used herein to describe the minimal amountof target DNA that must be present in a sample in order for detection tooccur. Thus, as an illustrative example, when a threshold of detectionis 10 nM, then a signal can be detected when a target DNA is present inthe sample at a concentration of 10 nM or more. In some cases, a methodof the present disclosure has a threshold of detection of 5 nM or less.In some cases, a method of the present disclosure has a threshold ofdetection of 1 nM or less. In some cases, a method of the presentdisclosure has a threshold of detection of 0.5 nM or less. In somecases, a method of the present disclosure has a threshold of detectionof 0.1 nM or less. In some cases, a method of the present disclosure hasa threshold of detection of 0.05 nM or less. In some cases, a method ofthe present disclosure has a threshold of detection of 0.01 nM or less.In some cases, a method of the present disclosure has a threshold ofdetection of 0.005 nM or less. In some cases, a method of the presentdisclosure has a threshold of detection of 0.001 nM or less. In somecases, a method of the present disclosure has a threshold of detectionof 0.0005 nM or less. In some cases, a method of the present disclosurehas a threshold of detection of 0.0001 nM or less. In some cases, amethod of the present disclosure has a threshold of detection of 0.00005nM or less. In some cases, a method of the present disclosure has athreshold of detection of 0.00001 nM or less. In some cases, a method ofthe present disclosure has a threshold of detection of 10 pM or less. Insome cases, a method of the present disclosure has a threshold ofdetection of 1 pM or less. In some cases, a method of the presentdisclosure has a threshold of detection of 500 fM or less. In somecases, a method of the present disclosure has a threshold of detectionof 250 fM or less. In some cases, a method of the present disclosure hasa threshold of detection of 100 fM or less. In some cases, a method ofthe present disclosure has a threshold of detection of 50 fM or less. Insome cases, a method of the present disclosure has a threshold ofdetection of 500 aM (attomolar) or less. In some cases, a method of thepresent disclosure has a threshold of detection of 250 aM or less. Insome cases, a method of the present disclosure has a threshold ofdetection of 100 aM or less. In some cases, a method of the presentdisclosure has a threshold of detection of 50 aM or less. In some cases,a method of the present disclosure has a threshold of detection of 10 aMor less. In some cases, a method of the present disclosure has athreshold of detection of 1 aM or less.

In some cases, the threshold of detection (for detecting the target DNAin a subject method), is in a range of from 500 fM to 1 nM (e.g., from500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500fM to 10 pM, from 500 fM to 1 pM, from 800 fM to 1 nM, from 800 fM to500 pM, from 800 fM to 200 pM, from 800 fM to 100 pM, from 800 fM to 10pM, from 800 fM to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1pM to 200 pM, from 1 pM to 100 pM, or from 1 pM to 10 pM) (where theconcentration refers to the threshold concentration of target DNA atwhich the target DNA can be detected). In some cases, a method of thepresent disclosure has a threshold of detection in a range of from 800fM to 100 pM. In some cases, a method of the present disclosure has athreshold of detection in a range of from 1 pM to 10 pM. In some cases,a method of the present disclosure has a threshold of detection in arange of from 10 fM to 500 fM, e.g., from 10 fM to 50 fM, from 50 fM to100 fM, from 100 fM to 250 fM, or from 250 fM to 500 fM.

In some cases, the minimum concentration at which a target DNA can bedetected in a sample is in a range of from 500 fM to 1 nM (e.g., from500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500fM to 10 pM, from 500 fM to 1 pM, from 800 fM to 1 nM, from 800 fM to500 pM, from 800 fM to 200 pM, from 800 fM to 100 pM, from 800 fM to 10pM, from 800 fM to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1pM to 200 pM, from 1 pM to 100 pM, or from 1 pM to 10 pM). In somecases, the minimum concentration at which a target DNA can be detectedin a sample is in a range of from 800 fM to 100 pM. In some cases, theminimum concentration at which a target DNA can be detected in a sampleis in a range of from 1 pM to 10 pM.

In some cases, the threshold of detection (for detecting the target DNAin a subject method), is in a range of from 1 aM to 1 nM (e.g., from 1aM to 500 pM, from 1 aM to 200 pM, from 1 aM to 100 pM, from 1 aM to 10pM, from 1 aM to 1 pM, from 100 aM to 1 nM, from 100 aM to 500 pM, from100 aM to 200 pM, from 100 aM to 100 pM, from 100 aM to 10 pM, from 100aM to 1 pM, from 250 aM to 1 nM, from 250 aM to 500 pM, from 250 aM to200 pM, from 250 aM to 100 pM, from 250 aM to 10 pM, from 250 aM to 1pM, from 500 aM to 1 nM, from 500 aM to 500 pM, from 500 aM to 200 pM,from 500 aM to 100 pM, from 500 aM to 10 pM, from 500 aM to 1 pM, from750 aM to 1 nM, from 750 aM to 500 pM, from 750 aM to 200 pM, from 750aM to 100 pM, from 750 aM to 10 pM, from 750 aM to 1 pM, from 1 fM to 1nM, from 1 fM to 500 pM, from 1 fM to 200 pM, from 1 fM to 100 pM, from1 fM to 10 pM, from 1 fM to 1 pM, from 500 fM to 500 pM, from 500 fM to200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 1pM, from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM,from 800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1pM to 1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100pM, or from 1 pM to 10 pM) (where the concentration refers to thethreshold concentration of target DNA at which the target DNA can bedetected). In some cases, a method of the present disclosure has athreshold of detection in a range of from 1 aM to 800 aM. In some cases,a method of the present disclosure has a threshold of detection in arange of from 50 aM to 1 pM. In some cases, a method of the presentdisclosure has a threshold of detection in a range of from 50 aM to 500fM.

In some cases, the minimum concentration at which a target DNA can bedetected in a sample is in a range of from 1 aM to 1 nM (e.g., from 1 aMto 500 pM, from 1 aM to 200 pM, from 1 aM to 100 pM, from 1 aM to 10 pM,from 1 aM to 1 pM, from 100 aM to 1 nM, from 100 aM to 500 pM, from 100aM to 200 pM, from 100 aM to 100 pM, from 100 aM to 10 pM, from 100 aMto 1 pM, from 250 aM to 1 nM, from 250 aM to 500 pM, from 250 aM to 200pM, from 250 aM to 100 pM, from 250 aM to 10 pM, from 250 aM to 1 pM,from 500 aM to 1 nM, from 500 aM to 500 pM, from 500 aM to 200 pM, from500 aM to 100 pM, from 500 aM to 10 pM, from 500 aM to 1 pM, from 750 aMto 1 nM, from 750 aM to 500 pM, from 750 aM to 200 pM, from 750 aM to100 pM, from 750 aM to 10 pM, from 750 aM to 1 pM, from 1 fM to 1 nM,from 1 fM to 500 pM, from 1 fM to 200 pM, from 1 fM to 100 pM, from 1 fMto 10 pM, from 1 fM to 1 pM, from 500 fM to 500 pM, from 500 fM to 200pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 1 pM,from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM, from800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1 pMto 1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100 pM,or from 1 pM to 10 pM). In some cases, the minimum concentration atwhich a target DNA can be detected in a sample is in a range of from 1aM to 500 pM. In some cases, the minimum concentration at which a targetDNA can be detected in a sample is in a range of from 100 aM to 500 pM.

In some cases, a subject composition or method exhibits an attomolar(aM) sensitivity of detection. In some cases, a subject composition ormethod exhibits a femtomolar (fM) sensitivity of detection. In somecases, a subject composition or method exhibits a picomolar (pM)sensitivity of detection. In some cases, a subject composition or methodexhibits a nanomolar (nM) sensitivity of detection.

Target DNA

A target DNA can be single stranded (ssDNA) or double stranded (dsDNA).When the target DNA is single stranded, there is no preference orrequirement for a PAM sequence in the target DNA. However, when thetarget DNA is dsDNA, a PAM is usually present adjacent to the targetsequence of the target DNA (e.g., see discussion of the PAM elsewhereherein). The source of the target DNA can be the same as the source ofthe sample, e.g., as described below.

The source of the target DNA can be any source. In some cases, thetarget DNA is a viral DNA (e.g., a genomic DNA of a DNA virus). As such,subject method can be for detecting the presence of a viral DNA amongsta population of nucleic acids (e.g., in a sample). A subject method canalso be used for the cleavage of non-target ssDNAs in the present of atarget DNA. For example, if a method takes place in a cell, a subjectmethod can be used to promiscuously cleave non-target ssDNAs in the cell(ssDNAs that do not hybridize with the guide sequence of the guide RNA)when a particular target DNA is present in the cell (e.g., when the cellis infected with a virus and viral target DNA is detected).

Examples of possible target DNAs include, but are not limited to, viralDNAs such as: a papovavirus (e.g., human papillomavirus (HPV),polyomavirus); a hepadnavirus (e.g., Hepatitis B Virus (HBV)); aherpesvirus (e.g., herpes simplex virus (HSV), varicella zoster virus(VZV), epstein-barr virus (EBV), cytomegalovirus (CMV), herpeslymphotropic virus, Pityriasis Rosea, kaposi's sarcoma-associatedherpesvirus); an adenovirus (e.g., atadenovirus, aviadenovirus,ichtadenovirus, mastadenovirus, siadenovirus); a poxvirus (e.g.,smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus,pseudocowpox, bovine papular stomatitis virus; tanapox virus, yabamonkey tumor virus; molluscum contagiosum virus (MCV)); a parvovirus(e.g., adeno-associated virus (AAV), Parvovirus B19, human bocavirus,bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; Phycodnaviridae;and the like. In some cases, the target DNA is parasite DNA. In somecases, the target DNA is bacterial DNA, e.g., DNA of a pathogenicbacterium.

Samples

A subject sample includes nucleic acid (e.g., a plurality of nucleicacids). The term “plurality” is used herein to mean two or more. Thus,in some cases, a sample includes two or more (e.g., 3 or more, 5 ormore, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more,1,000 or more, or 5,000 or more) nucleic acids (e.g., DNAs). A subjectmethod can be used as a very sensitive way to detect a target DNApresent in a sample (e.g., in a complex mixture of nucleic acids such asDNAs). In some cases, the sample includes 5 or more DNAs (e.g., 10 ormore, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more,or 5,000 or more DNAs) that differ from one another in sequence. In somecases, the sample includes 10 or more, 20 or more, 50 or more, 100 ormore, 500 or more, 10³ or more, 5×10³ or more, 10⁴ or more, 5×10⁴ ormore, 10⁵ or more, 5×10⁵ or more, 10⁶ or more 5×10⁶ or more, or 10⁷ ormore, DNAs. In some cases, the sample comprises from 10 to 20, from 20to 50, from 50 to 100, from 100 to 500, from 500 to 10³, from 10³ to5×10³, from 5×10³ to 10⁴, from 10⁴ to 5×10⁴, from 5×10⁴ to 10⁵, from 10⁵to 5×10⁵, from 5×10⁵ to 10⁶, from 10⁶ to 5×10⁶, or from 5×10⁶ to 10⁷, ormore than 10⁷, DNAs. In some cases, the sample comprises from 5 to 10⁷DNAs (e.g., that differ from one another in sequence)(e.g., from 5 to10⁶, from 5 to 10⁵, from 5 to 50,000, from 5 to 30,000, from 10 to 10⁶,from 10 to 10⁵, from 10 to 50,000, from 10 to 30,000, from 20 to 10⁶,from 20 to 10⁵, from 20 to 50,000, or from 20 to 30,000 DNAs). In somecases, the sample includes 20 or more DNAs that differ from one anotherin sequence. In some cases, the sample includes DNAs from a cell lysate(e.g., a eukaryotic cell lysate, a mammalian cell lysate, a human celllysate, a prokaryotic cell lysate, a plant cell lysate, and the like).For example, in some cases the sample includes DNA from a cell such as aeukaryotic cell, e.g., a mammalian cell such as a human cell.

The term “sample” is used herein to mean any sample that includes DNA(e.g., in order to determine whether a target DNA is present among apopulation of DNAs). The sample can be derived from any source, e.g.,the sample can be a synthetic combination of purified DNAs; the samplecan be a cell lysate, an DNA-enriched cell lysate, or DNAs isolatedand/or purified from a cell lysate. The sample can be from a patient(e.g., for the purpose of diagnosis). The sample can be frompermeabilized cells. The sample can be from crosslinked cells. Thesample can be in tissue sections. The sample can be from tissuesprepared by crosslinking followed by delipidation and adjustment to makea uniform refractive index. Examples of tissue preparation bycrosslinking followed by delipidation and adjustment to make a uniformrefractive index have been described in, for example, Shah et al.,Development (2016) 143, 2862-2867 doi:10.1242/dev.138560.

A “sample” can include a target DNA and a plurality of non-target DNAs.In some cases, the target DNA is present in the sample at one copy per10 non-target DNAs, one copy per 20 non-target DNAs, one copy per 25non-target DNAs, one copy per 50 non-target DNAs, one copy per 100non-target DNAs, one copy per 500 non-target DNAs, one copy per 10³non-target DNAs, one copy per 5×10³ non-target DNAs, one copy per 10⁴non-target DNAs, one copy per 5×10⁴ non-target DNAs, one copy per 10⁵non-target DNAs, one copy per 5×10⁵ non-target DNAs, one copy per 10⁶non-target DNAs, or less than one copy per 10⁶ non-target DNAs. In somecases, the target DNA is present in the sample at from one copy per 10non-target DNAs to 1 copy per 20 non-target DNAs, from 1 copy per 20non-target DNAs to 1 copy per 50 non-target DNAs, from 1 copy per 50non-target DNAs to 1 copy per 100 non-target DNAs, from 1 copy per 100non-target DNAs to 1 copy per 500 non-target DNAs, from 1 copy per 500non-target DNAs to 1 copy per 10³ non-target DNAs, from 1 copy per 10³non-target DNAs to 1 copy per 5×10³ non-target DNAs, from 1 copy per5×10³ non-target DNAs to 1 copy per 10⁴ non-target DNAs, from 1 copy per10⁴ non-target DNAs to 1 copy per 10⁵ non-target DNAs, from 1 copy per10⁵ non-target DNAs to 1 copy per 10⁶ non-target DNAs, or from 1 copyper 10⁶ non-target DNAs to 1 copy per 10⁷ non-target DNAs.

Suitable samples include but are not limited to saliva, blood, serum,plasma, urine, aspirate, and biopsy samples. Thus, the term “sample”with respect to a patient encompasses blood and other liquid samples ofbiological origin, solid tissue samples such as a biopsy specimen ortissue cultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents; washed; orenrichment for certain cell populations, such as cancer cells. Thedefinition also includes sample that have been enriched for particulartypes of molecules, e.g., DNAs. The term “sample” encompasses biologicalsamples such as a clinical sample such as blood, plasma, serum,aspirate, cerebral spinal fluid (CSF), and also includes tissue obtainedby surgical resection, tissue obtained by biopsy, cells in culture, cellsupernatants, cell lysates, tissue samples, organs, bone marrow, and thelike. A “biological sample” includes biological fluids derived therefrom(e.g., cancerous cell, infected cell, etc.), e.g., a sample comprisingDNAs that is obtained from such cells (e.g., a cell lysate or other cellextract comprising DNAs).

A sample can comprise, or can be obtained from, any of a variety ofcells, tissues, organs, or acellular fluids. Suitable sample sourcesinclude eukaryotic cells, bacterial cells, and archaeal cells. Suitablesample sources include single-celled organisms and multi-cellularorganisms. Suitable sample sources include single-cell eukaryoticorganisms; a plant or a plant cell; an algal cell, e.g., Botryococcusbraunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorellapyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell(e.g., a yeast cell); an animal cell, tissue, or organ; a cell, tissue,or organ from an invertebrate animal (e.g. fruit fly, cnidarian,echinoderm, nematode, an insect, an arachnid, etc.); a cell, tissue,fluid, or organ from a vertebrate animal (e.g., fish, amphibian,reptile, bird, mammal); a cell, tissue, fluid, or organ from a mammal(e.g., a human; a non-human primate; an ungulate; a feline; a bovine; anovine; a caprine; etc.). Suitable sample sources include nematodes,protozoans, and the like. Suitable sample sources include parasites suchas helminths, malarial parasites, etc.

Suitable sample sources include a cell, tissue, or organism of any ofthe six kingdoms, e.g., Bacteria (e.g., Eubacteria); Archaebacteria;Protista; Fungi; Plantae; and Animalia. Suitable sample sources includeplant-like members of the kingdom Protista, including, but not limitedto, algae (e.g., green algae, red algae, glaucophytes, cyanobacteria);fungus-like members of Protista, e.g., slime molds, water molds, etc;animal-like members of Protista, e.g., flagellates (e.g., Euglena),amoeboids (e.g., amoeba), sporozoans (e.g, Apicomplexa, Myxozoa,Microsporidia), and ciliates (e.g., Paramecium). Suitable sample sourcesinclude members of the kingdom Fungi, including, but not limited to,members of any of the phyla: Basidiomycota (club fungi; e.g., members ofAgaricus, Amanita, Boletus, Cantherellus, etc.); Ascomycota (sac fungi,including, e.g., Saccharomyces); Mycophycophyta (lichens); Zygomycota(conjugation fungi); and Deuteromycota. Suitable sample sources includemembers of the kingdom Plantae, including, but not limited to, membersof any of the following divisions: Bryophyta (e.g., mosses),Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g., liverworts),Lycophyta (e.g., club mosses), Sphenophyta (e.g., horsetails),Psilophyta (e.g., whisk ferns), Ophioglossophyta, Pterophyta (e.g.,ferns), Cycadophyta, Gingkophyta, Pinophyta, Gnetophyta, andMagnoliophyta (e.g., flowering plants). Suitable sample sources includemembers of the kingdom Animalia, including, but not limited to, membersof any of the following phyla: Porifera (sponges); Placozoa;Orthonectida (parasites of marine invertebrates); Rhombozoa; Cnidaria(corals, anemones, jellyfish, sea pens, sea pansies, sea wasps);Ctenophora (comb jellies); Platyhelminthes (flatworms); Nemertina(ribbon worms); Ngathostomulida (jawed worms)p Gastrotricha; Rotifera;Priapulida; Kinorhyncha; Loricifera; Acanthocephala; Entoprocta;Nemotoda; Nematomorpha; Cycliophora; Mollusca (mollusks); Sipuncula(peanut worms); Annelida (segmented worms); Tardigrada (water bears);Onychophora (velvet worms); Arthropoda (including the subphyla:Chelicerata, Myriapoda, Hexapoda, and Crustacea, where the Cheliceratainclude, e.g., arachnids, Merostomata, and Pycnogonida, where theMyriapoda include, e.g., Chilopoda (centipedes), Diplopoda (millipedes),Paropoda, and Symphyla, where the Hexapoda include insects, and wherethe Crustacea include shrimp, hill, barnacles, etc.; Phoronida;Ectoprocta (moss animals); Brachiopoda; Echinodermata (e.g. starfish,sea daisies, feather stars, sea urchins, sea cucumbers, brittle stars,brittle baskets, etc.); Chaetognatha (arrow worms); Hemichordata (acornworms); and Chordata. Suitable members of Chordata include any member ofthe following subphyla: Urochordata (sea squirts; including Ascidiacea,Thaliacea, and Larvacea); Cephalochordata (lancelets); Myxini (hagfish);and Vertebrata, where members of Vertebrata include, e.g., members ofPetromyzontida (lampreys), Chondrichthyces (cartilaginous fish),Actinopterygii (ray-finned fish), Actinista (coelocanths), Dipnoi(lungfish), Reptilia (reptiles, e.g., snakes, alligators, crocodiles,lizards, etc.), Ayes (birds); and Mammalian (mammals) Suitable plantsinclude any monocotyledon and any dicotyledon.

Suitable sources of a sample include cells, fluid, tissue, or organtaken from an organism; from a particular cell or group of cellsisolated from an organism; etc. For example, where the organism is aplant, suitable sources include xylem, the phloem, the cambium layer,leaves, roots, etc. Where the organism is an animal, suitable sourcesinclude particular tissues (e.g., lung, liver, heart, kidney, brain,spleen, skin, fetal tissue, etc.), or a particular cell type (e.g.,neuronal cells, epithelial cells, endothelial cells, astrocytes,macrophages, glial cells, islet cells, T lymphocytes, B lymphocytes,etc.).

In some cases, the source of the sample is a (or is suspected of being adiseased cell, fluid, tissue, or organ. In some cases, the source of thesample is a normal (non-diseased) cell, fluid, tissue, or organ. In somecases, the source of the sample is a (or is suspected of being) apathogen-infected cell, tissue, or organ. For example, the source of asample can be an individual who may or may not be infected—and thesample could be any biological sample (e.g., blood, saliva, biopsy,plasma, serum, bronchoalveolar lavage, sputum, a fecal sample,cerebrospinal fluid, a fine needle aspirate, a swab sample (e.g., abuccal swab, a cervical swab, a nasal swab), interstitial fluid,synovial fluid, nasal discharge, tears, buffy coat, a mucous membranesample, an epithelial cell sample (e.g., epithelial cell scraping),etc.) collected from the individual. In some cases, the sample is acell-free liquid sample. In some cases, the sample is a liquid samplethat can comprise cells. Pathogens include viruses, fungi, helminths,protozoa, malarial parasites, Plasmodium parasites, Toxoplasmaparasites, Schistosoma parasites, and the like. “Helminths” includeroundworms, heartworms, and phytophagous nematodes (Nematoda), flukes(Tematoda), Acanthocephala, and tapeworms (Cestoda). Protozoaninfections include infections from Giardia spp., Trichomonas spp.,African trypanosomiasis, amoebic dysentery, babesiosis, balantidialdysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis.Examples of pathogens such as parasitic/protozoan pathogens include, butare not limited to: Plasmodium falciparum, Plasmodium vivax, Trypanosomacruzi and Toxoplasma gondii. Fungal pathogens include, but are notlimited to: Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis,and Candida albicans. Pathogenic viruses include, e.g., humanimmunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nilevirus; herpes virus; yellow fever virus; Hepatitis C Virus; Hepatitis AVirus; Hepatitis B Virus; papillomavirus; and the like. Pathogenicviruses can include DNA viruses such as: a papovavirus (e.g., humanpapillomavirus (HPV), polyomavirus); a hepadnavirus (e.g., Hepatitis BVirus (HBV)); a herpesvirus (e.g., herpes simplex virus (HSV), varicellazoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV),herpes lymphotropic virus, Pityriasis Rosea, Kaposi's sarcoma-associatedherpesvirus); an adenovirus (e.g., atadenovirus, aviadenovirus,ichtadenovirus, mastadenovirus, siadenovirus); a poxvirus (e.g.,smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus,pseudocowpox, bovine papular stomatitis virus; tanapox virus, yabamonkey tumor virus; molluscum contagiosum virus (MCV)); a parvovirus(e.g., adeno-associated virus (AAV), Parvovirus B19, human bocavirus,bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; Phycodnaviridae;and the like. Pathogens can include, e.g., DNAviruses (e.g.: apapovavirus (e.g., human papillomavirus (HPV), polyomavirus); ahepadnavirus (e.g., Hepatitis B Virus (HBV)); a herpesvirus (e.g.,herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barrvirus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus,Pityriasis Rosea, Kaposi's sarcoma-associated herpesvirus); anadenovirus (e.g., atadenovirus, aviadenovirus, ichtadenovirus,mastadenovirus, siadenovirus); a poxvirus (e.g., smallpox, vacciniavirus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovinepapular stomatitis virus; tanapox virus, yaba monkey tumor virus;molluscum contagiosum virus (MCV)); a parvovirus (e.g., adeno-associatedvirus (AAV), Parvovirus B19, human bocavirus, bufavirus, human parv4G1); Geminiviridae; Nanoviridae; Phycodnaviridae; and the like],Mycobacterium tuberculosis, Streptococcus agalactiae,methicillin-resistant Staphylococcus aureus, Legionella pneumophila,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans,Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpessimplex virus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, Reovirus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, West Nile virus, Plasmodiumfalciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli,Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei,Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeriatenella, Onchocerca volvulus, Leishmania tropica, Mycobacteriumtuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena,Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoidescorti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae.

Measuring a Detectable Signal

In some cases, a subject method includes a step of measuring (e.g.,measuring a detectable signal produced by Cas12J-mediated ssDNAcleavage). Because a Cas12J polypeptide of the present disclosurecleaves non-targeted ssDNA once activated, which occurs when a guide RNAhybridizes with a target DNA in the presence of a Cas12J effectorprotein, a detectable signal can be any signal that is produced whenssDNA is cleaved. For example, in some cases, the step of measuring caninclude one or more of: gold nanoparticle based detection (e.g., see Xuet al., Angew Chem Int Ed Engl. 2007; 46(19):3468-70; and Xia et al.,Proc Natl Acad Sci USA. 2010 Jun. 15; 107(24):10837-41), fluorescencepolarization, colloid phase transition/dispersion (e.g., Baksh et al.,Nature. 2004 Jan. 8; 427(6970):139-41), electrochemical detection,semiconductor-based sensing (e.g., Rothberg et al., Nature. 2011 Jul.20; 475(7356):348-52; e.g., one could use a phosphatase to generate a pHchange after ssDNA cleavage reactions, by opening 2′-3′ cyclicphosphates, and by releasing inorganic phosphate into solution), anddetection of a labeled detector ssDNA (see elsewhere herein for moredetails). The readout of such detection methods can be any convenientreadout. Examples of possible readouts include but are not limited to: ameasured amount of detectable fluorescent signal; a visual analysis ofbands on a gel (e.g., bands that represent cleaved product versusuncleaved substrate), a visual or sensor based detection of the presenceor absence of a color (i.e., color detection method), and the presenceor absence of (or a particular amount of) an electrical signal.

The measuring can in some cases be quantitative, e.g., in the sense thatthe amount of signal detected can be used to determine the amount oftarget DNA present in the sample. The measuring can in some cases bequalitative, e.g., in the sense that the presence or absence ofdetectable signal can indicate the presence or absence of targeted DNA(e.g., virus, SNP, etc.). In some cases, a detectable signal will not bepresent (e.g., above a given threshold level) unless the targeted DNA(s)(e.g., virus, SNP, etc.) is present above a particular thresholdconcentration. In some cases, the threshold of detection can be titratedby modifying the amount of Cas12J effector, guide RNA, sample volume,and/or detector ssDNA (if one is used). As such, for example, as wouldbe understood by one of ordinary skill in the art, a number of controlscan be used if desired in order to set up one or more reactions, eachset up to detect a different threshold level of target DNA, and thussuch a series of reactions could be used to determine the amount oftarget DNA present in a sample (e.g., one could use such a series ofreactions to determine that a target DNA is present in the sample ‘at aconcentration of at least X’).

Examples of uses of a detection method of the present disclosureinclude, e.g., single nucleotide polymorphism (SNP) detection, cancerscreening, detection of bacterial infection, detection of antibioticresistance, detection of viral infection, and the like. The compositionsand methods of this disclosure can be used to detect any DNA target. Forexample, any virus that integrates nucleic acid material into the genomecan be detected because a subject sample can include cellular genomicDNA—and the guide RNA can be designed to detect integrated nucleotidesequence.

In some cases, a method of the present disclosure can be used todetermine the amount of a target DNA in a sample (e.g., a samplecomprising the target DNA and a plurality of non-target DNAs).Determining the amount of a target DNA in a sample can comprisecomparing the amount of detectable signal generated from a test sampleto the amount of detectable signal generated from a reference sample.Determining the amount of a target DNA in a sample can comprise:measuring the detectable signal to generate a test measurement;measuring a detectable signal produced by a reference sample to generatea reference measurement; and comparing the test measurement to thereference measurement to determine an amount of target DNA present inthe sample.

For example, in some cases, a method of the present disclosure fordetermining the amount of a target DNA in a sample comprises: a)contacting the sample (e.g., a sample comprising the target DNA and aplurality of non-target DNAs) with: (i) a guide RNA that hybridizes withthe target DNA, (ii) a Cas12J polypeptide of the present disclosure thatcleaves RNAs present in the sample, and (iii) a detector ssDNA; b)measuring a detectable signal produced by Cas12J-mediated ssDNA cleavage(e.g., cleavage of the detector ssDNA), generating a test measurement;c) measuring a detectable signal produced by a reference sample togenerate a reference measurement; and d) comparing the test measurementto the reference measurement to determine an amount of target DNApresent in the sample.

As another example, in some cases, a method of the present disclosurefor determining the amount of a target DNA in a sample comprises: a)contacting the sample (e.g., a sample comprising the target DNA and aplurality of non-target DNAs) with: i) a precursor guide RNA arraycomprising two or more guide RNAs each of which has a different guidesequence; (ii) a Cas12J polypeptide of the present disclosure thatcleaves the precursor guide RNA array into individual guide RNAs, andalso cleaves RNAs of the sample; and (iii) a detector ssDNA; b)measuring a detectable signal produced by Cas12J-mediated ssDNA cleavage(e.g., cleavage of the detector ssDNA), generating a test measurement;c) measuring a detectable signal produced by each of two or morereference samples to generate two or more reference measurements; and d)comparing the test measurement to the reference measurements todetermine an amount of target DNA present in the sample.

Amplification of Nucleic Acids in the Sample

In some embodiments, sensitivity of a subject composition and/or method(e.g., for detecting the presence of a target DNA, such as viral DNA ora SNP, in cellular genomic DNA) can be increased by coupling detectionwith nucleic acid amplification. In some cases, the nucleic acids in asample are amplified prior to contact with a Cas12J polypeptide of thepresent disclosure that cleaved ssDNA (e.g., amplification of nucleicacids in the sample can begin prior to contact with a Cas12J polypeptideof the present disclosure). In some cases, the nucleic acids in a sampleare amplified simultaneously with contact with a Cas12J polypeptide ofthe present disclosure. For example, in some cases, a subject methodincludes amplifying nucleic acids of a sample (e.g., by contacting thesample with amplification components) prior to contacting the amplifiedsample with a Cas12J polypeptide of the present disclosure. In somecases, a subject method includes contacting a sample with amplificationcomponents at the same time (simultaneous with) that the sample iscontacted with a Cas12J polypeptide of the present disclosure. If allcomponents are added simultaneously (amplification components anddetection components such as a Cas12J polypeptide of the presentdisclosure, a guide RNA, and a detector DNA), it is possible that thetrans-cleavage activity of the Cas12J will begin to degrade the nucleicacids of the sample at the same time the nucleic acids are undergoingamplification. However, even if this is the case, amplifying anddetecting simultaneously can still increase sensitivity compared toperforming the method without amplification.

In some cases, specific sequences (e.g., sequences of a virus, sequencesthat include a SNP of interest) are amplified from the sample, e.g.,using primers. As such, a sequence to which the guide RNA will hybridizecan be amplified in order to increase sensitivity of a subject detectionmethod—this could achieve biased amplification of a desired sequence inorder to increase the number of copies of the sequence of interestpresent in the sample relative to other sequences present in the sample.As one illustrative example, if a subject method is being used todetermine whether a given sample includes a particular virus (or aparticular SNP), a desired region of viral sequence (or non-viralgenomic sequence) can be amplified, and the region amplified willinclude the sequence that would hybridize to the guide RNA if the viralsequence (or SNP) were in fact present in the sample.

As noted, in some cases the nucleic acids are amplified (e.g., bycontact with amplification components) prior to contacting the amplifiednucleic acids with a Cas12J polypeptide of the present disclosure. Insome cases, amplification occurs for 10 seconds or more, (e.g., 30seconds or more, 45 seconds or more, 1 minute or more, 2 minutes ormore, 3 minutes or more, 4 minutes or more, 5 minutes or more, 7.5minutes or more, 10 minutes or more, etc.) prior to contact with aCas12J polypeptide of the present disclosure. In some cases,amplification occurs for 2 minutes or more (e.g., 3 minutes or more, 4minutes or more, 5 minutes or more, 7.5 minutes or more, 10 minutes ormore, etc.) prior to contact with a Cas12J polypeptide of the presentdisclosure. In some cases, amplification occurs for a period of time ina range of from 10 seconds to 60 minutes (e.g., 10 seconds to 40minutes, 10 seconds to 30 minutes, 10 seconds to 20 minutes, 10 secondsto 15 minutes, 10 seconds to 10 minutes, 10 seconds to 5 minutes, 30seconds to 40 minutes, 30 seconds to 30 minutes, 30 seconds to 20minutes, 30 seconds to 15 minutes, 30 seconds to 10 minutes, 30 secondsto 5 minutes, 1 minute to 40 minutes, 1 minute to 30 minutes, 1 minuteto 20 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, 1 minuteto 5 minutes, 2 minutes to 40 minutes, 2 minutes to 30 minutes, 2minutes to 20 minutes, 2 minutes to 15 minutes, 2 minutes to 10 minutes,2 minutes to 5 minutes, 5 minutes to 40 minutes, 5 minutes to 30minutes, 5 minutes to 20 minutes, 5 minutes to 15 minutes, or 5 minutesto 10 minutes). In some cases, amplification occurs for a period of timein a range of from 5 minutes to 15 minutes. In some cases, amplificationoccurs for a period of time in a range of from 7 minutes to 12 minutes.

In some cases, a sample is contacted with amplification components atthe same time as contact with a Cas12J polypeptide of the presentdisclosure. In some such cases, the Cas12J protein is inactive at thetime of contact and is activated once nucleic acids in the sample havebeen amplified.

Various amplification methods and components will be known to one ofordinary skill in the art and any convenient method can be used (see,e.g., Zanoli and Spoto, Biosensors (Basel). 2013 March; 3(1): 18-43;Gill and Ghaemi, Nucleosides, Nucleotides, and Nucleic Acids, 2008, 27:224-243; Craw and Balachandrana, Lab Chip, 2012, 12, 2469-2486; whichare herein incorporated by reference in their entirety). Nucleic acidamplification can comprise polymerase chain reaction (PCR), reversetranscription PCR (RT-PCR), quantitative PCR (qPCR), reversetranscription qPCR (RT-qPCR), nested PCR, multiplex PCR, asymmetric PCR,touchdown PCR, random primer PCR, hemi-nested PCR, polymerase cyclingassembly (PCA), colony PCR, ligase chain reaction (LCR), digital PCR,methylation specific-PCR (MSP), co-amplification at lower denaturationtemperature-PCR (COLD-PCR), allele-specific PCR, intersequence-specificPCR (ISS-PCR), whole genome amplification (WGA), inverse PCR, andthermal asymmetric interlaced PCR (TAIL-PCR).

In some cases, the amplification is isothermal amplification. The term“isothermal amplification” indicates a method of nucleic acid (e.g.,DNA) amplification (e.g., using enzymatic chain reaction) that can use asingle temperature incubation thereby obviating the need for a thermalcycler. Isothermal amplification is a form of nucleic acid amplificationwhich does not rely on the thermal denaturation of the target nucleicacid during the amplification reaction and hence may not requiremultiple rapid changes in temperature. Isothermal nucleic acidamplification methods can therefore be carried out inside or outside ofa laboratory environment. By combining with a reverse transcriptionstep, these amplification methods can be used to isothermally amplifyRNA.

Examples of isothermal amplification methods include but are not limitedto: loop-mediated isothermal Amplification (LAMP), helicase-dependentAmplification (HDA), recombinase polymerase amplification (RPA), stranddisplacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA), transcription mediated amplification (TMA),nicking enzyme amplification reaction (NEAR), rolling circleamplification (RCA), multiple displacement amplification (MDA),Ramification (RAM), circular helicase-dependent amplification (cHDA),single primer isothermal amplification (SPIA), signal mediatedamplification of RNA technology (SMART), self-sustained sequencereplication (3SR), genome exponential amplification reaction (GEAR) andisothermal multiple displacement amplification (IMDA).

In some cases, the amplification is recombinase polymerase amplification(RPA) (see, e.g., U.S. Pat. Nos. 8,030,000; 8,426,134; 8,945,845;9,309,502; and 9,663,820, which are hereby incorporated by reference intheir entirety). Recombinase polymerase amplification (RPA) uses twoopposing primers (much like PCR) and employs three enzymes—arecombinase, a single-stranded DNA-binding protein (SSB) and astrand-displacing polymerase. The recombinase pairs oligonucleotideprimers with homologous sequence in duplex DNA, SSB binds to displacedstrands of DNA to prevent the primers from being displaced, and thestrand displacing polymerase begins DNA synthesis where the primer hasbound to the target DNA. Adding a reverse transcriptase enzyme to an RPAreaction can facilitate detection RNA as well as DNA, without the needfor a separate step to produce cDNA. One example of components for anRPA reaction is as follows (see, e.g., U.S. Pat. Nos. 8,030,000;8,426,134; 8,945,845; 9,309,502; 9,663,820): 50 mM Tris pH 8.4, 80 mMPotassium acetate, 10 mM Magnesium acetate, 2 mM dithiothreitol (DTT),5% PEG compound (Carbowax-20M), 3 mM ATP, 30 mM Phosphocreatine, 100ng/μl creatine kinase, 420 ng/μl gp32, 140 ng/μl UvsX, 35 ng/μl UvsY,2000M dNTPs, 300 nM each oligonucleotide, 35 ng/μl Bsu polymerase, and anucleic acid-containing sample).

In a transcription mediated amplification (TMA), an RNA polymerase isused to make RNA from a promoter engineered in the primer region, andthen a reverse transcriptase synthesizes cDNA from the primer. A thirdenzyme, e.g., Rnase H can then be used to degrade the RNA target fromcDNA without the heat-denatured step. This amplification technique issimilar to Self-Sustained Sequence Replication (3SR) and Nucleic AcidSequence Based Amplification (NASBA), but varies in the enzymesemployed. For another example, helicase-dependent amplification (HDA)utilizes a thermostable helicase (Tte-UvrD) rather than heat to unwinddsDNA to create single-strands that are then available for hybridizationand extension of primers by polymerase. For yet another example, a loopmediated amplification (LAMP) employs a thermostable polymerase withstrand displacement capabilities and a set of four or more specificdesigned primers. Each primer is designed to have hairpin ends that,once displaced, snap into a hairpin to facilitate self-priming andfurther polymerase extension. In a LAMP reaction, though the reactionproceeds under isothermal conditions, an initial heat denaturation stepis required for double-stranded targets. In addition, amplificationyields a ladder pattern of various length products. For yet anotherexample, a strand displacement amplification (SDA) combines the abilityof a restriction endonuclease to nick the unmodified strand of itstarget DNA and an exonuclease-deficient DNA polymerase to extend the 3′end at the nick and displace the downstream DNA strand.

Detector DNA

In some cases, a subject method includes contacting a sample (e.g., asample comprising a target DNA and a plurality of non-target ssDNAs)with: i) a Cas12J polypeptide of the present disclosure; ii) a guide RNA(or precursor guide RNA array); and iii) a detector DNA that is singlestranded and does not hybridize with the guide sequence of the guideRNA. For example, in some cases, a subject method includes contacting asample with a labeled single stranded detector DNA (detector ssDNA) thatincludes a fluorescence-emitting dye pair; the Cas12J polypeptidecleaves the labeled detector ssDNA after it is activated (by binding tothe guide RNA in the context of the guide RNA hybridizing to a targetDNA); and the detectable signal that is measured is produced by thefluorescence-emitting dye pair. For example, in some cases, a subjectmethod includes contacting a sample with a labeled detector ssDNAcomprising a fluorescence resonance energy transfer (FRET) pair or aquencher/fluor pair, or both. In some cases, a subject method includescontacting a sample with a labeled detector ssDNA comprising a FRETpair. In some cases, a subject method includes contacting a sample witha labeled detector ssDNA comprising a fluor/quencher pair.

Fluorescence-emitting dye pairs comprise a FRET pair or a quencher/fluorpair. In both cases of a FRET pair and a quencher/fluor pair, theemission spectrum of one of the dyes overlaps a region of the absorptionspectrum of the other dye in the pair. As used herein, the term“fluorescence-emitting dye pair” is a generic term used to encompassboth a “fluorescence resonance energy transfer (FRET) pair” and a“quencher/fluor pair,” both of which terms are discussed in more detailbelow. The term “fluorescence-emitting dye pair” is used interchangeablywith the phrase “a FRET pair and/or a quencher/fluor pair.”

In some cases (e.g., when the detector ssDNA includes a FRET pair) thelabeled detector ssDNA produces an amount of detectable signal prior tobeing cleaved, and the amount of detectable signal that is measured isreduced when the labeled detector ssDNA is cleaved. In some cases, thelabeled detector ssDNA produces a first detectable signal prior to beingcleaved (e.g., from a FRET pair) and a second detectable signal when thelabeled detector ssDNA is cleaved (e.g., from a quencher/fluor pair). Assuch, in some cases, the labeled detector ssDNA comprises a FRET pairand a quencher/fluor pair.

In some cases, the labeled detector ssDNA comprises a FRET pair. FRET isa process by which radiationless transfer of energy occurs from anexcited state fluorophore to a second chromophore in close proximity.The range over which the energy transfer can take place is limited toapproximately 10 nanometers (100 angstroms), and the efficiency oftransfer is extremely sensitive to the separation distance betweenfluorophores. Thus, as used herein, the term “FRET” (“fluorescenceresonance energy transfer”; also known as “Förster resonance energytransfer”) refers to a physical phenomenon involving a donor fluorophoreand a matching acceptor fluorophore selected so that the emissionspectrum of the donor overlaps the excitation spectrum of the acceptor,and further selected so that when donor and acceptor are in closeproximity (usually 10 nm or less) to one another, excitation of thedonor will cause excitation of and emission from the acceptor, as someof the energy passes from donor to acceptor via a quantum couplingeffect. Thus, a FRET signal serves as a proximity gauge of the donor andacceptor; only when they are in close proximity to one another is asignal generated. The FRET donor moiety (e.g., donor fluorophore) andFRET acceptor moiety (e.g., acceptor fluorophore) are collectivelyreferred to herein as a “FRET pair”.

The donor-acceptor pair (a FRET donor moiety and a FRET acceptor moiety)is referred to herein as a “FRET pair” or a “signal FRET pair.” Thus, insome cases, a subject labeled detector ssDNA includes two signalpartners (a signal pair), when one signal partner is a FRET donor moietyand the other signal partner is a FRET acceptor moiety. A subjectlabeled detector ssDNA that includes such a FRET pair (a FRET donormoiety and a FRET acceptor moiety) will thus exhibit a detectable signal(a FRET signal) when the signal partners are in close proximity (e.g.,while on the same RNA molecule), but the signal will be reduced (orabsent) when the partners are separated (e.g., after cleavage of the RNAmolecule by a Cas12J polypeptide of the present disclosure).

FRET donor and acceptor moieties (FRET pairs) will be known to one ofordinary skill in the art and any convenient FRET pair (e.g., anyconvenient donor and acceptor moiety pair) can be used. Examples ofsuitable FRET pairs include but are not limited to those presented inTable 1. See also: Bajar et al. Sensors (Basel). 2016 Sep. 14; 16(9);and Abraham et al. PLoS One. 2015 Aug. 3; 10(8):e0134436.

TABLE 1 Examples of FRET pairs (donor and acceptor FRET moieties) DonorAcceptor Tryptophan Dansyl IAEDANS (1) DDPM (2) BFP DsRFP DansylFluorescein isothiocyanate (FITC) Dansyl Octadecylrhodamine Cyanfluorescent Green fluorescent protein protein (CFP) (GFP) CF (3) TexasRed Fluorescein Tetramethylrhodamine Cy3 Cy5 GFP Yellow fluorescentprotein (YFP) BODIPY FL (4) BODIPY FL (4) Rhodamine 110 Cy3 Rhodamine 6GMalachite Green FITC Eosin Thiosemicarbazide B-Phycoerythrin Cy5 Cy5Cy5.5 (1) 5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid (2)N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (3) carboxyfluoresceinsuccinimidyl ester (4) 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

In some cases, a detectable signal is produced when the labeled detectorssDNA is cleaved (e.g., in some cases, the labeled detector ssDNAcomprises a quencher/fluor pair). One signal partner of a signalquenching pair produces a detectable signal and the other signal partneris a quencher moiety that quenches the detectable signal of the firstsignal partner (i.e., the quencher moiety quenches the signal of thesignal moiety such that the signal from the signal moiety is reduced(quenched) when the signal partners are in proximity to one another,e.g., when the signal partners of the signal pair are in closeproximity).

For example, in some cases, an amount of detectable signal increaseswhen the labeled detector ssDNA is cleaved. For example, in some cases,the signal exhibited by one signal partner (a signal moiety) is quenchedby the other signal partner (a quencher signal moiety), e.g., when bothare present on the same ssDNA molecule prior to cleavage by a Cas12Jpolypeptide of the present disclosure). Such a signal pair is referredto herein as a “quencher/fluor pair”, “quenching pair”, or “signalquenching pair.” For example, in some cases, one signal partner (e.g.,the first signal partner) is a signal moiety that produces a detectablesignal that is quenched by the second signal partner (e.g., a quenchermoiety). The signal partners of such a quencher/fluor pair will thusproduce a detectable signal when the partners are separated (e.g., aftercleavage of the detector ssDNA by a Cas12J polypeptide of the presentdisclosure), but the signal will be quenched when the partners are inclose proximity (e.g., prior to cleavage of the detector ssDNA by aCas12J polypeptide of the present disclosure).

A quencher moiety can quench a signal from the signal moiety (e.g.,prior to cleave of the detector ssDNA by a Cas12J polypeptide of thepresent disclosure) to various degrees. In some cases, a quencher moietyquenches the signal from the signal moiety where the signal detected inthe presence of the quencher moiety (when the signal partners are inproximity to one another) is 95% or less of the signal detected in theabsence of the quencher moiety (when the signal partners are separated).For example, in some cases, the signal detected in the presence of thequencher moiety can be 90% or less, 80% or less, 70% or less, 60% orless, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less,10% or less, or 5% or less of the signal detected in the absence of thequencher moiety. In some cases, no signal (e.g., above background) isdetected in the presence of the quencher moiety.

In some cases, the signal detected in the absence of the quencher moiety(when the signal partners are separated) is at least 1.2 fold greater(e.g., at least 1.3 fold, at least 1.5 fold, at least 1.7 fold, at least2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 20fold, or at least 50 fold greater) than the signal detected in thepresence of the quencher moiety (when the signal partners are inproximity to one another).

In some cases, the signal moiety is a fluorescent label. In some suchcases, the quencher moiety quenches the signal (the light signal) fromthe fluorescent label (e.g., by absorbing energy in the emission spectraof the label). Thus, when the quencher moiety is not in proximity withthe signal moiety, the emission (the signal) from the fluorescent labelis detectable because the signal is not absorbed by the quencher moiety.Any convenient donor acceptor pair (signal moiety/quencher moiety pair)can be used and many suitable pairs are known in the art.

In some cases, the quencher moiety absorbs energy from the signal moiety(also referred to herein as a “detectable label”) and then emits asignal (e.g., light at a different wavelength). Thus, in some cases, thequencher moiety is itself a signal moiety (e.g., a signal moiety can be6-carboxyfluorescein while the quencher moiety can be6-carboxy-tetramethylrhodamine), and in some such cases, the pair couldalso be a FRET pair. In some cases, a quencher moiety is a darkquencher. A dark quencher can absorb excitation energy and dissipate theenergy in a different way (e.g., as heat). Thus, a dark quencher hasminimal to no fluorescence of its own (does not emit fluorescence).Examples of dark quenchers are further described in U.S. Pat. Nos.8,822,673 and 8,586,718; U.S. patent publications 20140378330,20140349295, and 20140194611; and international patent applications:WO200142505 and WO200186001, all if which are hereby incorporated byreference in their entirety.

Examples of fluorescent labels include, but are not limited to: an AlexaFluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488,ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550,ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101,ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3,Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy dye,a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, a Square dye,fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), TexasRed, Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, quantumdots, and a tethered fluorescent protein.

In some cases, a detectable label is a fluorescent label selected from:an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465,ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542,ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12,ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTORho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665,ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye(e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye,a Sulfo Cy dye, a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, aSquare dye, fluorescein (FITC), tetramethylrhodamine (TRITC), Texas Red,Oregon Green, Pacific Blue, Pacific Green, and Pacific Orange.

In some cases, a detectable label is a fluorescent label selected from:an Alexa Fluor® dye, an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465,ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542,ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12,ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTORho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665,ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye(e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye,a Sulfo Cy dye, a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, aSquare dye, fluorescein (FITC), tetramethylrhodamine (TRITC), Texas Red,Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, a quantumdot, and a tethered fluorescent protein.

Examples of ATTO dyes include, but are not limited to: ATTO 390, ATTO425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTORho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12,ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12,ATTO 665, ATTO 680, ATTO 700, ATTO 725, and ATTO 740.

Examples of AlexaFluor dyes include, but are not limited to: AlexaFluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, AlexaFluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, AlexaFluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, AlexaFluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, AlexaFluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Alexa Fluor® 790, andthe like.

Examples of quencher moieties include, but are not limited to: a darkquencher, a Black Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2,BHQ-3), a Qx1 quencher, an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q,and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (Dabsyl), IowaBlack RQ, Iowa Black FQ, IRDye QC-1, a QSY dye (e.g., QSY 7, QSY 9, QSY21), AbsoluteQuencher, Eclipse, and metal clusters such as goldnanoparticles, and the like.

In some cases, a quencher moiety is selected from: a dark quencher, aBlack Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), a Qx1quencher, an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q),dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa BlackFQ, IRDye QC-1, a QSY dye (e.g., QSY 7, QSY 9, QSY 21),AbsoluteQuencher, Eclipse, and a metal cluster.

Examples of an ATTO quencher include, but are not limited to: ATTO 540Q,ATTO 580Q, and ATTO 612Q. Examples of a Black Hole Quencher® (BHQ®)include, but are not limited to: BHQ-0 (493 nm), BHQ-1 (534 nm), BHQ-2(579 nm) and BHQ-3 (672 nm).

For examples of some detectable labels (e.g., fluorescent dyes) and/orquencher moieties, see, e.g., Bao et al., Annu Rev Biomed Eng. 2009;11:25-47; as well as U.S. Pat. Nos. 8,822,673 and 8,586,718; U.S. patentpublications 20140378330, 20140349295, 20140194611, 20130323851,20130224871, 20110223677, 20110190486, 20110172420, 20060179585 and20030003486; and international patent applications: WO200142505 andWO200186001, all of which are hereby incorporated by reference in theirentirety.

In some cases, cleavage of a labeled detector ssDNA can be detected bymeasuring a colorimetric read-out. For example, the liberation of afluorophore (e.g., liberation from a FRET pair, liberation from aquencher/fluor pair, and the like) can result in a wavelength shift (andthus color shift) of a detectable signal. Thus, in some cases, cleavageof a subject labeled detector ssDNA can be detected by a color-shift.Such a shift can be expressed as a loss of an amount of signal of onecolor (wavelength), a gain in the amount of another color, a change inthe ration of one color to another, and the like.

Transgenic, Non-Human Organisms

As described above, in some cases, a nucleic acid (e.g., a recombinantexpression vector) of the present disclosure (e.g., a nucleic acidcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure; a nucleic acid comprising a nucleotide sequenceencoding a Cas12J fusion polypeptide of the present disclosure; etc.),is used as a transgene to generate a transgenic non-human organism thatproduces a Cas12J polypeptide, or a Cas12J fusion polypeptide, of thepresent disclosure. The present disclosure provides atransgenic-non-human organism comprising a nucleotide sequence encodinga Cas12J polypeptide, or a Cas12J fusion polypeptide, of the presentdisclosure.

Transgenic, Non-Human Animals

The present disclosure provides a transgenic non-human animal, whichanimal comprises a transgene comprising a nucleic acid comprising anucleotide sequence encoding a Cas12J polypeptide or a Cas12J fusionpolypeptide. In some embodiments, the genome of the transgenic non-humananimal comprises a nucleotide sequence encoding a Cas12J polypeptide ora Cas12J fusion polypeptide, of the present disclosure. In some cases,the transgenic non-human animal is homozygous for the geneticmodification. In some cases, the transgenic non-human animal isheterozygous for the genetic modification. In some embodiments, thetransgenic non-human animal is a vertebrate, for example, a fish (e.g.,salmon, trout, zebra fish, gold fish, puffer fish, cave fish, etc.), anamphibian (frog, newt, salamander, etc.), a bird (e.g., chicken, turkey,etc.), a reptile (e.g., snake, lizard, etc.), a non-human mammal (e.g.,an ungulate, e.g., a pig, a cow, a goat, a sheep, etc.; a lagomorph(e.g., a rabbit); a rodent (e.g., a rat, a mouse); a non-human primate;etc.), etc. In some cases, the transgenic non-human animal is aninvertebrate. In some cases, the transgenic non-human animal is aninsect (e.g., a mosquito; an agricultural pest; etc.). In some cases,the transgenic non-human animal is an arachnid.

Nucleotide sequences encoding a Cas12J polypeptide, or a Cas12J fusionpolypeptide, of the present disclosure can be under the control of(i.e., operably linked to) an unknown promoter (e.g., when the nucleicacid randomly integrates into a host cell genome) or can be under thecontrol of (i.e., operably linked to) a known promoter. Suitable knownpromoters can be any known promoter and include constitutively activepromoters (e.g., CMV promoter), inducible promoters (e.g., heat shockpromoter, tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.),spatially restricted and/or temporally restricted promoters (e.g., atissue specific promoter, a cell type specific promoter, etc.), etc.

Transgenic Plants

As described above, in some cases, a nucleic acid (e.g., a recombinantexpression vector) of the present disclosure (e.g., a nucleic acidcomprising a nucleotide sequence encoding a Cas12J polypeptide of thepresent disclosure; a nucleic acid comprising a nucleotide sequenceencoding a Cas12J fusion polypeptide of the present disclosure; etc.),is used as a transgene to generate a transgenic plant that produces aCas12J polypeptide, or a Cas12J fusion polypeptide, of the presentdisclosure. The present disclosure provides a transgenic plantcomprising a nucleotide sequence encoding a Cas12J polypeptide, or aCas12J fusion polypeptide, of the present disclosure. In someembodiments, the genome of the transgenic plant comprises a subjectnucleic acid. In some embodiments, the transgenic plant is homozygousfor the genetic modification. In some embodiments, the transgenic plantis heterozygous for the genetic modification.

Methods of introducing exogenous nucleic acids into plant cells are wellknown in the art. Such plant cells are considered “transformed,” asdefined above. Suitable methods include viral infection (such as doublestranded DNA viruses), transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calcium phosphateprecipitation, direct microinjection, silicon carbide whiskerstechnology, Agrobacterium-mediated transformation and the like. Thechoice of method is generally dependent on the type of cell beingtransformed and the circumstances under which the transformation istaking place (i.e. in vitro, ex vivo, or in vivo).

Transformation methods based upon the soil bacterium Agrobacteriumtumefaciens are particularly useful for introducing an exogenous nucleicacid molecule into a vascular plant. The wild type form of Agrobacteriumcontains a Ti (tumor-inducing) plasmid that directs production oftumorigenic crown gall growth on host plants. Transfer of thetumor-inducing T-DNA region of the Ti plasmid to a plant genome requiresthe Ti plasmid-encoded virulence genes as well as T-DNA borders, whichare a set of direct DNA repeats that delineate the region to betransferred. An Agrobacterium-based vector is a modified form of a Tiplasmid, in which the tumor inducing functions are replaced by thenucleic acid sequence of interest to be introduced into the plant host.

Agrobacterium-mediated transformation generally employs cointegratevectors or binary vector systems, in which the components of the Tiplasmid are divided between a helper vector, which resides permanentlyin the Agrobacterium host and carries the virulence genes, and a shuttlevector, which contains the gene of interest bounded by T-DNA sequences.A variety of binary vectors is well known in the art and arecommercially available, for example, from Clontech (Palo Alto, Calif.).Methods of coculturing Agrobacterium with cultured plant cells orwounded tissue such as leaf tissue, root explants, hypocotyledons, stempieces or tubers, for example, also are well known in the art. See,e.g., Glick and Thompson, (eds.), Methods in Plant Molecular Biology andBiotechnology, Boca Raton, Fla.: CRC Press (1993).

Microprojectile-mediated transformation also can be used to produce asubject transgenic plant. This method, first described by Klein et al.(Nature 327:70-73 (1987)), relies on microprojectiles such as gold ortungsten that are coated with the desired nucleic acid molecule byprecipitation with calcium chloride, spermidine or polyethylene glycol.The microprojectile particles are accelerated at high speed into anangiosperm tissue using a device such as the BIOLISTIC PD-1000 (Biorad;Hercules Calif.).

A nucleic acid of the present disclosure (e.g., a nucleic acid (e.g., arecombinant expression vector) comprising a nucleotide sequence encodinga Cas12J polypeptide, or a Cas12J fusion polypeptide, of the presentdisclosure) may be introduced into a plant in a manner such that thenucleic acid is able to enter a plant cell(s), e.g., via an in vivo orex vivo protocol. By “in vivo,” it is meant in the nucleic acid isadministered to a living body of a plant e.g. infiltration. By “ex vivo”it is meant that cells or explants are modified outside of the plant,and then such cells or organs are regenerated to a plant. A number ofvectors suitable for stable transformation of plant cells or for theestablishment of transgenic plants have been described, including thosedescribed in Weissbach and Weissbach, (1989) Methods for Plant MolecularBiology Academic Press, and Gelvin et al., (1990) Plant MolecularBiology Manual, Kluwer Academic Publishers. Specific examples includethose derived from a Ti plasmid of Agrobacterium tumefaciens, as well asthose disclosed by Herrera-Estrella et al. (1983) Nature 303: 209, Bevan(1984) Nucl Acid Res. 12: 8711-8721, Klee (1985) Bio/Technolo 3:637-642. Alternatively, non-Ti vectors can be used to transfer the DNAinto plants and cells by using free DNA delivery techniques. By usingthese methods transgenic plants such as wheat, rice (Christou (1991)Bio/Technology 9:957-9 and 4462) and corn (Gordon-Kamm (1990) Plant Cell2: 603-618) can be produced. An immature embryo can also be a goodtarget tissue for monocots for direct DNA delivery techniques by usingthe particle gun (Weeks et al. (1993) Plant Physiol 102: 1077-1084;Vasil (1993) Bio/Technolo 10: 667-674; Wan and Lemeaux (1994) PlantPhysiol 104: 37-48 and for Agrobacterium-mediated DNA transfer (Ishidaet al. (1996) Nature Biotech 14: 745-750). Exemplary methods forintroduction of DNA into chloroplasts are biolistic bombardment,polyethylene glycol transformation of protoplasts, and microinjection(Danieli et al Nat. Biotechnol 16:345-348, 1998; Staub et al Nat.Biotechnol 18: 333-338, 2000; O'Neill et al Plant J. 3:729-738, 1993;Knoblauch et al Nat. Biotechnol 17: 906-909; U.S. Pat. Nos. 5,451,513,5,545,817, 5,545,818, and 5,576,198; in Intl. Application No. WO95/16783; and in Boynton et al., Methods in Enzymology 217: 510-536(1993), Svab et al., Proc. Natl. Acad. Sci. USA 90: 913-917 (1993), andMcBride et al., Proc. Natl. Acad. Sci. USA 91: 7301-7305 (1994)). Anyvector suitable for the methods of biolistic bombardment, polyethyleneglycol transformation of protoplasts and microinjection will be suitableas a targeting vector for chloroplast transformation. Any doublestranded DNA vector may be used as a transformation vector, especiallywhen the method of introduction does not utilize Agrobacterium.

Plants which can be genetically modified include grains, forage crops,fruits, vegetables, oil seed crops, palms, forestry, and vines. Specificexamples of plants which can be modified follow: maize, banana, peanut,field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats,potato, soybeans, cotton, carnations, sorghum, lupin and rice.

The present disclosure provides transformed plant cells, tissues, plantsand products that contain the transformed plant cells. A feature of thesubject transformed cells, and tissues and products that include thesame is the presence of a subject nucleic acid integrated into thegenome, and production by plant cells of a Cas12J polypeptide, or aCas12J fusion polypeptide, of the present disclosure. Recombinant plantcells of the present invention are useful as populations of recombinantcells, or as a tissue, seed, whole plant, stem, fruit, leaf, root,flower, stem, tuber, grain, animal feed, a field of plants, and thelike.

Nucleotide sequences encoding a Cas12J polypeptide, or a Cas12J fusionpolypeptide, of the present disclosure can be under the control of(i.e., operably linked to) an unknown promoter (e.g., when the nucleicacid randomly integrates into a host cell genome) or can be under thecontrol of (i.e., operably linked to) a known promoter. Suitable knownpromoters can be any known promoter and include constitutively activepromoters, inducible promoters, spatially restricted and/or temporallyrestricted promoters, etc.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-149 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

Aspect 1. A composition comprising: a) a Cas12J polypeptide, or anucleic acid molecule encoding the Cas12J polypeptide; and b) a Cas12Jguide RNA, or one or more DNA molecules encoding the Cas12J guide RNA.

Aspect 2. The composition of aspect 1, wherein the Cas12J polypeptidecomprises an amino acid sequence having 50% or more amino acid sequenceidentity to the amino acid sequence depicted in any one of FIG. 6A-6R.

Aspect 3. The composition of aspect 1 or aspect 2, wherein the Cas12Jguide RNA comprises a nucleotide sequence having 80%, 90%, 95%, 98%,99%, or 100%, nucleotide sequence identity with any one of the crRNAsequences depicted in FIG. 7.

Aspect 4. The composition of aspect 1 or aspect 2, wherein the Cas12Jpolypeptide is fused to a nuclear localization signal (NLS).

Aspect 5. The composition of any one of aspects 1-4, wherein thecomposition comprises a lipid.

Aspect 6. The composition of any one of aspects 1-4, wherein a) and b)are within a liposome.

Aspect 7. The composition of any one of aspects 1-4, wherein a) and b)are within a particle.

Aspect 8. The composition of any one of aspects 1-7, comprising one ormore of: a buffer, a nuclease inhibitor, and a protease inhibitor.

Aspect 9. The composition of any one of aspects 1-8, wherein the Cas12Jpolypeptide comprises an amino acid sequence having 85% or more identityto the amino acid sequence depicted in any one of FIG. 6A-6R.

Aspect 10. The composition of any one of aspects 1-9, wherein the Cas12Jpolypeptide is a nickase that can cleave only one strand of adouble-stranded target nucleic acid molecule.

Aspect 11. The composition of any one of aspects 1-9, wherein the Cas12Jpolypeptide is a catalytically inactive Cas12J polypeptide (dCas12J).

Aspect 12. The composition of aspect 10 or aspect 11, wherein the Cas12Jpolypeptide comprises one or more mutations at a position correspondingto those selected from: D464, E678, and D769 of Cas12J_10037042_3.

Aspect 13. The composition of any one of aspects 1-12, furthercomprising a DNA donor template.

Aspect 14. A Cas12J fusion polypeptide comprising: a Cas12J polypeptidefused to a heterologous polypeptide.

Aspect 15. The Cas12J fusion polypeptide of Aspect 14, wherein theCas12J polypeptide comprises an amino acid sequence having 50% or moreidentity to the amino acid sequence depicted in any one of FIG. 6A-6R.

Aspect 16. The Cas12J fusion polypeptide of Aspect 14, wherein theCas12J polypeptide comprises an amino acid sequence having 85% or moreidentity to the amino acid sequence depicted in any one of FIG. 6A-6R.

Aspect 17. The Cas12J fusion polypeptide of any one of aspects 14-16,wherein the Cas12J polypeptide is a nickase that can cleave only onestrand of a double-stranded target nucleic acid molecule.

Aspect 18. The Cas12J fusion polypeptide of any one of aspects 14-17,wherein the Cas12J polypeptide is a catalytically inactive Cas12Jpolypeptide (dCas12J).

Aspect 19. The Cas12J fusion polypeptide of aspect 17 or aspect 18,wherein the Cas12J polypeptide comprises one or more mutations at aposition corresponding to those selected from: D464, E678, and D769 ofCas12J_10037042_3.

Aspect 20. The Cas12J fusion polypeptide of any one of aspects 14-19,wherein the heterologous polypeptide is fused to the N-terminus and/orthe C-terminus of the Cas12J polypeptide.

Aspect 21. The Cas12J fusion polypeptide of any one of aspects 14-20,comprising a nuclear localization signal (NLS).

Aspect 22. The Cas12J fusion polypeptide of any one of aspects 14-21,wherein the heterologous polypeptide is a targeting polypeptide thatprovides for binding to a cell surface moiety on a target cell or targetcell type.

Aspect 23. The Cas12J fusion polypeptide of any one of aspects 14-21,wherein the heterologous polypeptide exhibits an enzymatic activity thatmodifies target DNA.

Aspect 24. The Cas12J fusion polypeptide of aspect 23, wherein theheterologous polypeptide exhibits one or more enzymatic activitiesselected from: nuclease activity, methyltransferase activity,demethylase activity, DNA repair activity, DNA damage activity,deamination activity, dismutase activity, alkylation activity,depurination activity, oxidation activity, pyrimidine dimer formingactivity, integrase activity, transposase activity, recombinaseactivity, polymerase activity, ligase activity, helicase activity,photolyase activity and glycosylase activity.

Aspect 25. The Cas12J fusion polypeptide of aspect 24, wherein theheterologous polypeptide exhibits one or more enzymatic activitiesselected from: nuclease activity, methyltransferase activity,demethylase activity, deamination activity, depurination activity,integrase activity, transposase activity, and recombinase activity.

Aspect 26. The Cas12J fusion polypeptide of any one of aspects 14-21,wherein the heterologous polypeptide exhibits an enzymatic activity thatmodifies a target polypeptide associated with a target nucleic acid.

Aspect 27. The Cas12J fusion polypeptide of aspect 26, wherein theheterologous polypeptide exhibits histone modification activity.

Aspect 28. The Cas12J fusion polypeptide of aspect 26 or aspect 27,wherein the heterologous polypeptide exhibits one or more enzymaticactivities selected from: methyltransferase activity, demethylaseactivity, acetyltransferase activity, deacetylase activity, kinaseactivity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity,SUMOylating activity, deSUMOylating activity, ribosylation activity,deribosylation activity, myristoylation activity, demyristoylationactivity, glycosylation activity (e.g., from O-GlcNAc transferase) anddeglycosylation activity.

Aspect 29. The Cas12J fusion polypeptide of aspect 28, wherein theheterologous polypeptide exhibits one or more enzymatic activitiesselected from: methyltransferase activity, demethylase activity,acetyltransferase activity, and deacetylase activity.

Aspect 30. The Cas12J fusion polypeptide of any one of aspects 14-21,wherein the heterologous polypeptide is an endosomal escape polypeptide.

Aspect 31. The Cas12J fusion polypeptide of aspect 30, wherein theendosomal escape polypeptide comprises an amino acid sequence selectedfrom: GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 36), and GLFHALLHLLHSLWHLLLHA(SEQ ID NO: 37), wherein each X is independently selected from lysine,histidine, and arginine.

Aspect 32. The Cas12J fusion polypeptide of any one of aspects 14-21,wherein the heterologous polypeptide is a chloroplast transit peptide.

Aspect 33. The Cas12J fusion polypeptide of any one of aspects 14-21,wherein the heterologous polypeptide comprises a protein transductiondomain.

Aspect 34. The Cas12J fusion polypeptide of any one of aspects 14-21,wherein the heterologous polypeptide is a protein that increases ordecreases transcription.

Aspect 35. The Cas12J fusion polypeptide of aspect 34, wherein theheterologous polypeptide is a transcriptional repressor domain.

Aspect 36. The Cas12J fusion polypeptide of aspect 34, wherein theheterologous polypeptide is a transcriptional activation domain.

Aspect 37. The Cas12J fusion polypeptide of any one of aspects 14-21,wherein the heterologous polypeptide is a protein binding domain.

Aspect 38. A nucleic acid comprising a nucleotide sequence encoding theCas12J fusion polypeptide of any one of aspects 14-37.

Aspect 39. The nucleic acid of Aspect 38, wherein the nucleotidesequence encoding the Cas12J fusion polypeptide is operably linked to apromoter.

Aspect 40. The nucleic acid of Aspect 39, wherein the promoter isfunctional in a eukaryotic cell.

Aspect 41. The nucleic acid of Aspect 40, wherein the promoter isfunctional in one or more of: a plant cell, a fungal cell, an animalcell, cell of an invertebrate, a fly cell, a cell of a vertebrate, amammalian cell, a primate cell, a non-human primate cell, and a humancell.

Aspect 43. The nucleic acid of any one of Aspects 39-41, wherein thepromoter is one or more of: a constitutive promoter, an induciblepromoter, a cell type-specific promoter, and a tissue-specific promoter.

Aspect 43. The nucleic acid of any one of Aspects 38-42, wherein thenucleic acid is a recombinant expression vector.

Aspect 44. The nucleic acid of Aspect 43, wherein the recombinantexpression vector is a recombinant adenoassociated viral vector, arecombinant retroviral vector, or a recombinant lentiviral vector.

Aspect 45. The nucleic acid of Aspect 39, wherein the promoter isfunctional in a prokaryotic cell.

Aspect 46. The nucleic acid of Aspect 38, wherein the nucleic acidmolecule is an mRNA.

Aspect 47. One or more nucleic acids comprising: (a) a nucleotidesequence encoding a Cas12J guide RNA; and (b) a nucleotide sequenceencoding a Cas12J polypeptide.

Aspect 48. The one or more nucleic acids of aspect 47, wherein theCas12J polypeptide comprises an amino acid sequence having 50% or moreidentity to the amino acid sequence depicted in any one of FIG. 6A-6R.

Aspect 49. The one or more nucleic acids of aspect 47, wherein theCas12J polypeptide comprises an amino acid sequence having 85% or moreidentity to the amino acid depicted in any one of FIG. 6A-6R.

Aspect 50. The one or more nucleic acids of any one of aspects 47-49,wherein the Cas12J guide RNA comprises a nucleotide sequence having 80%or more nucleotide sequence identity with any one of the crRNA sequencesset forth in FIG. 7.

Aspect 51. The one or more nucleic acids of any one of aspects 47-50,wherein the Cas12J polypeptide is fused to a nuclear localization signal(NLS).

Aspect 52. The one or more nucleic acids of any one of aspects 47-51,wherein the nucleotide sequence encoding the Cas12J guide RNA isoperably linked to a promoter.

Aspect 53. The one or more nucleic acids of any one of aspects 47-52,wherein the nucleotide sequence encoding the Cas12J polypeptide isoperably linked to a promoter.

Aspect 54. The one or more nucleic acids of Aspect 52 or Aspect 53,wherein the promoter operably linked to the nucleotide sequence encodingthe Cas12J guide RNA, and/or the promoter operably linked to thenucleotide sequence encoding the Cas12J polypeptide, is functional in aeukaryotic cell.

Aspect 55. The one or more nucleic acids of Aspect 54, wherein thepromoter is functional in one or more of: a plant cell, a fungal cell,an animal cell, cell of an invertebrate, a fly cell, a cell of avertebrate, a mammalian cell, a primate cell, a non-human primate cell,and a human cell.

Aspect 56. The one or more nucleic acids of any one of Aspects 53-55,wherein the promoter is one or more of: a constitutive promoter, aninducible promoter, a cell type-specific promoter, and a tissue-specificpromoter.

Aspect 57. The one or more nucleic acids of any one of Aspects 47-56,wherein the one or more nucleic acids is one or more recombinantexpression vectors.

Aspect 58. The one or more nucleic acids of Aspect 57, wherein the oneor more recombinant expression vectors are selected from: one or moreadenoassociated viral vectors, one or more recombinant retroviralvectors, or one or more recombinant lentiviral vectors.

Aspect 59. The one or more nucleic acids of Aspect 53, wherein thepromoter is functional in a prokaryotic cell.

Aspect 60. A eukaryotic cell comprising one or more of: a) a Cas12Jpolypeptide, or a nucleic acid comprising a nucleotide sequence encodingthe Cas12J polypeptide, b) a Cas12J fusion polypeptide, or a nucleicacid comprising a nucleotide sequence encoding the Cas12J fusionpolypeptide, and c) a Cas12J guide RNA, or a nucleic acid comprising anucleotide sequence encoding the Cas12J guide RNA.

Aspect 61. The eukaryotic cell of aspect 60, comprising the nucleic acidencoding the Cas12J polypeptide, wherein said nucleic acid is integratedinto the genomic DNA of the cell.

Aspect 62. The eukaryotic cell of aspect 60 or aspect 61, wherein theeukaryotic cell is a plant cell, a mammalian cell, an insect cell, anarachnid cell, a fungal cell, a bird cell, a reptile cell, an amphibiancell, an invertebrate cell, a mouse cell, a rat cell, a primate cell, anon-human primate cell, or a human cell.

Aspect 63. A cell comprising a comprising a Cas12J fusion polypeptide,or a nucleic acid comprising a nucleotide sequence encoding the Cas12Jfusion polypeptide.

Aspect 64. The cell of aspect 63, wherein the cell is a prokaryoticcell.

Aspect 65. The cell of aspect 63 or aspect 64, comprising the nucleicacid comprising a nucleotide sequence encoding the Cas12J fusionpolypeptide, wherein said nucleic acid molecule is integrated into thegenomic DNA of the cell.

Aspect 66. A method of modifying a target nucleic acid, the methodcomprising contacting the target nucleic acid with: a) a Cas12Jpolypeptide; and b) a Cas12J guide RNA comprising a guide sequence thathybridizes to a target sequence of the target nucleic acid, wherein saidcontacting results in modification of the target nucleic acid by theCas12J polypeptide.

Aspect 67. The method of aspect 66, wherein said modification iscleavage of the target nucleic acid.

Aspect 68. The method of aspect 66 or aspect 67, wherein the targetnucleic acid is selected from: double stranded DNA, single stranded DNA,RNA, genomic DNA, and extrachromosomal DNA.

Aspect 69. The method of any of aspects 66-68, wherein said contactingtakes place in vitro outside of a cell.

Aspect 70. The method of any of aspects 66-68, wherein said contactingtakes place inside of a cell in culture.

Aspect 71. The method of any of aspects 66-68, wherein said contactingtakes place inside of a cell in vivo.

Aspect 72. The method of aspect 70 or aspect 71, wherein the cell is aeukaryotic cell.

Aspect 73. The method of aspect 72, wherein the cell is selected from: aplant cell, a fungal cell, a mammalian cell, a reptile cell, an insectcell, an avian cell, a fish cell, a parasite cell, an arthropod cell, acell of an invertebrate, a cell of a vertebrate, a rodent cell, a mousecell, a rat cell, a primate cell, a non-human primate cell, and a humancell.

Aspect 74. The method of aspect 70 or aspect 71, wherein the cell is aprokaryotic cell.

Aspect 75. The method of any one of aspects 66-74, wherein saidcontacting results in genome editing.

Aspect 76. The method of any one of aspects 66-75, wherein saidcontacting comprises: introducing into a cell: (a) the Cas12Jpolypeptide, or a nucleic acid comprising a nucleotide sequence encodingthe Cas12J polypeptide, and (b) the Cas12J guide RNA, or a nucleic acidcomprising a nucleotide sequence encoding the Cas12J guide RNA.

Aspect 77. The method of aspect 76, wherein said contacting furthercomprises: introducing a DNA donor template into the cell.

Aspect 78. The method of any one of aspects 66-77, wherein the Cas12Jguide RNA comprises a nucleotide sequence having 80% or more nucleotidesequence identity with any one of the crRNA sequences set forth in FIG.7.

Aspect 79. The method of any one of aspects 66-78, wherein the Cas12Jpolypeptide is fused to a nuclear localization signal.

Aspect 80. A method of modulating transcription from a target DNA,modifying a target nucleic acid, or modifying a protein associated witha target nucleic acid, the method comprising contacting the targetnucleic acid with: a) a Cas12J fusion polypeptide comprising a Cas12Jpolypeptide fused to a heterologous polypeptide; and b) a Cas12J guideRNA comprising a guide sequence that hybridizes to a target sequence ofthe target nucleic acid.

Aspect 81. The method of aspect 80, wherein the Cas12J guide RNAcomprises a nucleotide sequence having 80% or more nucleotide sequenceidentity with any one of the crRNA sequences set forth in FIG. 7.

Aspect 82. The method of aspect 80 or aspect 81, wherein the Cas12Jfusion polypeptide comprises nuclear localization signal.

Aspect 83. The method of any of aspects 80-82, wherein said modificationis not cleavage of the target nucleic acid.

Aspect 84. The method of any of aspects 80-83, wherein the targetnucleic acid is selected from: double stranded DNA, single stranded DNA,RNA, genomic DNA, and extrachromosomal DNA.

Aspect 85. The method of any of aspects 80-84, wherein said contactingtakes place in vitro outside of a cell.

Aspect 86. The method of any of aspects 80-84, wherein said contactingtakes place inside of a cell in culture.

Aspect 87. The method of any of aspects 80-84, wherein said contactingtakes place inside of a cell in vivo.

Aspect 88. The method of aspect 86 or aspect 87, wherein the cell is aeukaryotic cell.

Aspect 89. The method of aspect 88, wherein the cell is selected from: aplant cell, a fungal cell, a mammalian cell, a reptile cell, an insectcell, an avian cell, a fish cell, a parasite cell, an arthropod cell, acell of an invertebrate, a cell of a vertebrate, a rodent cell, a mousecell, a rat cell, a primate cell, a non-human primate cell, and a humancell.

Aspect 90. The method of aspect 86 or aspect 87, wherein the cell is aprokaryotic cell.

Aspect 91. The method of any one of aspects 80-90, wherein saidcontacting comprises: introducing into a cell: (a) the Cas12J fusionpolypeptide, or a nucleic acid comprising a nucleotide sequence encodingthe Cas12J fusion polypeptide, and (b) the Cas12J guide RNA, or anucleic acid comprising a nucleotide sequence encoding the Cas12J guideRNA.

Aspect 92. The method of any one of aspects 80-91, wherein the Cas12Jpolypeptide is a catalytically inactive Cas12J polypeptide (dCas12J).

Aspect 93. The method of any one of aspects 80-92, wherein the Cas12Jpolypeptide comprises one or more amino acid substitutions at a positioncorresponding to those selected from: D464, E678, and D769 ofCas12J_10037042_3.

Aspect 94. The method of any one of aspects 80-93, wherein theheterologous polypeptide exhibits an enzymatic activity that modifiestarget DNA.

Aspect 95. The method of aspect 94, wherein the heterologous polypeptideexhibits an one or more enzymatic activities selected from: nucleaseactivity, methyltransferase activity, demethylase activity, DNA repairactivity, DNA damage activity, deamination activity, dismutase activity,alkylation activity, depurination activity, oxidation activity,pyrimidine dimer forming activity, integrase activity, transposaseactivity, recombinase activity, polymerase activity, ligase activity,helicase activity, photolyase activity and glycosylase activity.

Aspect 96. The method of aspect 95, wherein the heterologous polypeptideexhibits one or more enzymatic activities selected from: nucleaseactivity, methyltransferase activity, demethylase activity, deaminationactivity, depurination activity, integrase activity, transposaseactivity, and recombinase activity.

Aspect 97. The method of any one of aspects 80-93, wherein theheterologous polypeptide exhibits an enzymatic activity that modifies atarget polypeptide associated with a target nucleic acid.

Aspect 98. The method of aspect 97, wherein the heterologous polypeptideexhibits histone modification activity.

Aspect 99. The method of aspect 97 or aspect 98, wherein theheterologous polypeptide exhibits an one or more enzymatic activitiesselected from: methyltransferase activity, demethylase activity,acetyltransferase activity, deacetylase activity, kinase activity,phosphatase activity, ubiquitin ligase activity, deubiquitinatingactivity, adenylation activity, deadenylation activity, SUMOylatingactivity, deSUMOylating activity, ribosylation activity, deribosylationactivity, myristoylation activity, demyristoylation activity,glycosylation activity (e.g., from 0-GlcNAc transferase) anddeglycosylation activity.

Aspect 100. The method of aspect 99, wherein the heterologouspolypeptide exhibits one or more enzymatic activities selected from:methyltransferase activity, demethylase activity, acetyltransferaseactivity, and deacetylase activity.

Aspect 101. The method of any one of aspects 80-93, wherein theheterologous polypeptide is protein that increases or decreasestranscription.

Aspect 102. The method of aspect 101, wherein the heterologouspolypeptide is a transcriptional repressor domain.

Aspect 103. The method of aspect 101, wherein the heterologouspolypeptide is a transcriptional activation domain.

Aspect 104. The method of any one of aspects 80-93, wherein theheterologous polypeptide is a protein binding domain.

Aspect 105. A transgenic, multicellular, non-human organism whose genomecomprises a transgene comprising a nucleotide sequence encoding one ormore of: a) a Cas12J polypeptide; b) a Cas12J fusion polypeptide; and c)a Cas12J guide RNA

Aspect 106. The transgenic, multicellular, non-human organism of aspect105, wherein the Cas12J polypeptide comprises an amino acid sequencehaving 50% or more amino acid sequence identity to the amino acidsequence set forth in any one of FIG. 6A-6R.

Aspect 107. The transgenic, multicellular, non-human organism of aspect105, wherein the Cas12J polypeptide comprises an amino acid sequencehaving 85% or more amino acid sequence identity to the amino acidsequence set forth in any one of FIG. 6A-6R.

Aspect 108. The transgenic, multicellular, non-human organism of any oneof aspects 105-107, wherein the organism is a plant, a monocotyledonplant, a dicotyledon plant, an invertebrate animal, an insect, anarthropod, an arachnid, a parasite, a worm, a cnidarian, a vertebrateanimal, a fish, a reptile, an amphibian, an ungulate, a bird, a pig, ahorse, a sheep, a rodent, a mouse, a rat, or a non-human primate.

Aspect 109. A system comprising one of:

a) a Cas12J polypeptide and a Cas12J guide RNA;

b) a Cas12J polypeptide, a Cas12J guide RNA, and a DNA donor template;

c) a Cas12J fusion polypeptide and a Cas12J guide RNA;

d) a Cas12J fusion polypeptide, a Cas12J guide RNA, and a DNA donortemplate;

e) an mRNA encoding a Cas12J polypeptide, and a Cas12J guide RNA;

f) an mRNA encoding a Cas12J polypeptide; a Cas12J guide RNA, and a DNAdonor template;

g) an mRNA encoding a Cas12J fusion polypeptide, and a Cas12J guide RNA;

h) an mRNA encoding a Cas12J fusion polypeptide, a Cas12J guide RNA, anda DNA donor template;

i) one or more recombinant expression vectors comprising: i) anucleotide sequence encoding a Cas12J polypeptide; and ii) a nucleotidesequence encoding a Cas12J guide RNA;

j) one or more recombinant expression vectors comprising: i) anucleotide sequence encoding a Cas12J polypeptide; ii) a nucleotidesequence encoding a Cas12J guide RNA; and iii) a DNA donor template;

k) one or more recombinant expression vectors comprising: i) anucleotide sequence encoding a Cas12J fusion polypeptide; and ii) anucleotide sequence encoding a Cas12J guide RNA; and

l) one or more recombinant expression vectors comprising: i) anucleotide sequence encoding a Cas12J fusion polypeptide; ii) anucleotide sequence encoding a Cas12J guide RNA; and a DNA donortemplate.

Aspect 110. The Cas12J system of aspect 109, wherein the Cas12Jpolypeptide comprises an amino acid sequence having 50% or more aminoacid sequence identity to the amino acid sequence depicted in any one ofFIG. 6A-6R.

Aspect 111. The Cas12J system of aspect 109, wherein the Cas12Jpolypeptide comprises an amino acid sequence having 85% or more aminoacid sequence identity to the amino acid sequence depicted in any one ofFIG. 6A-6R.

Aspect 112. The Cas12J system of any of aspects 109-111, wherein thedonor template nucleic acid has a length of from 8 nucleotides to 1000nucleotides.

Aspect 113. The Cas12J system of any of aspects 109-111, wherein thedonor template nucleic acid has a length of from 25 nucleotides to 500nucleotides.

Aspect 114. A kit comprising the Cas12J system of any one of aspects109-113.

Aspect 115. The kit of aspect 114, wherein the components of the kit arein the same container.

Aspect 116. The kit of aspect 114, wherein the components of the kit arein separate containers.

Aspect 117. A sterile container comprising the Cas12J system of any oneof aspects 109-116.

Aspect 118. The sterile container of aspect 117, wherein the containeris a syringe.

Aspect 119. An implantable device comprising the Cas12J system of anyone of aspects 109-116.

Aspect 120. The implantable device of aspect 119, wherein the Cas12Jsystem is within a matrix.

Aspect 121. The implantable device of aspect 119, wherein the Cas12Jsystem is in a reservoir.

Aspect 122. A method of detecting a target DNA in a sample, the methodcomprising: (a) contacting the sample with: (i) a Cas12L polypeptide;(ii) a guide RNA comprising: a region that binds to the Cas12Lpolypeptide, and a guide sequence that hybridizes with the target DNA;and (iii) a detector DNA that is single stranded and does not hybridizewith the guide sequence of the guide RNA; and (b) measuring a detectablesignal produced by cleavage of the single stranded detector DNA by theCas12L polypeptide, thereby detecting the target DNA.

Aspect 123. The method of aspect 122, wherein the target DNA is singlestranded.

Aspect 124. The method of aspect 122, wherein the target DNA is doublestranded.

Aspect 125. The method of any one of aspects 122-124, wherein the targetDNA is bacterial DNA.

Aspect 126. The method of any one of aspects 122-124, wherein the targetDNA is viral DNA.

Aspect 127. The method of aspect 126, wherein the target DNA ispapovavirus, human papillomavirus (HPV), hepadnavirus, Hepatitis B Virus(HBV), herpesvirus, varicella zoster virus (VZV), Epstein-Barr virus(EBV), Kaposi's sarcoma-associated herpesvirus, adenovirus, poxvirus, orparvovirus DNA.

Aspect 128. The method of aspect 122, wherein the target DNA is from ahuman cell.

Aspect 129. The method of aspect 122, wherein the target DNA is humanfetal or cancer cell DNA.

Aspect 130. The method of any one of aspects 122-129, wherein the Cas12Jpolypeptide comprises an amino acid sequence having 50% or more aminoacid sequence identity to the amino acid sequence depicted in any one ofFIG. 6A-6R.

Aspect 131. The method of aspect 122, wherein the sample comprises DNAfrom a cell lysate.

Aspect 132. The method of aspect 122, wherein the sample comprisescells.

Aspect 133. The method of aspect 122, wherein the sample is a blood,serum, plasma, urine, aspirate, or biopsy sample.

Aspect 134. The method of any one of aspects 122-133, further comprisingdetermining an amount of the target DNA present in the sample.

Aspect 135. The method of aspect 122, wherein said measuring adetectable signal comprises one or more of: visual based detection,sensor-based detection, color detection, gold nanoparticle baseddetection, fluorescence polarization, colloid phasetransition/dispersion, electrochemical detection, andsemiconductor-based sensing.

Aspect 136. The method of any one of aspects 122-135, wherein thelabeled detector DNA comprises a modified nucleobase, a modified sugarmoiety, and/or a modified nucleic acid linkage.

Aspect 137. The method of any one of aspects 122-135, further comprisingdetecting a positive control target DNA in a positive control sample,the detecting comprising: (c) contacting the positive control samplewith: (i) the Cas12J polypeptide; (ii) a positive control guide RNAcomprising: a region that binds to the Cas12J polypeptide, and apositive control guide sequence that hybridizes with the positivecontrol target DNA; and (iii) a labeled detector DNA that is singlestranded and does not hybridize with the positive control guide sequenceof the positive control guide RNA; and (d) measuring a detectable signalproduced by cleavage of the labeled detector DNA by the Cas12Jpolypeptide, thereby detecting the positive control target DNA

Aspect 138. The method of any one of aspects 122-136, wherein thedetectable signal is detectable in less than 45 minutes.

Aspect 139. The method of any one of aspects 122-136, wherein thedetectable signal is detectable in less than 30 minutes.

Aspect 140. The method of any one of aspects 122-139, further comprisingamplifying the target DNA in the sample by loop-mediated isothermalamplification (LAMP), helicase-dependent amplification (HDA),recombinase polymerase amplification (RPA), strand displacementamplification (SDA), nucleic acid sequence-based amplification (NASBA),transcription mediated amplification (TMA), nicking enzyme amplificationreaction (NEAR), rolling circle amplification (RCA), multipledisplacement amplification (MDA), Ramification (RAM), circularhelicase-dependent amplification (cHDA), single primer isothermalamplification (SPIA), signal mediated amplification of RNA technology(SMART), self-sustained sequence replication (3SR), genome exponentialamplification reaction (GEAR), or isothermal multiple displacementamplification (IMDA).

Aspect 141. The method of any one of aspects 122-140, wherein target DNAin the sample is present at a concentration of less than 10 aM.

Aspect 142. The method according to any one of aspect 122-141, whereinthe single stranded detector DNA comprises a fluorescence-emitting dyepair.

Aspect 143. The method according to aspect 142, wherein thefluorescence-emitting dye pair produces an amount of detectable signalprior to cleavage of the single stranded detector DNA, and the amount ofdetectable signal is reduced after cleavage of the single strandeddetector DNA.

Aspect 144. The method according to aspect 142, wherein the singlestranded detector DNA produces a first detectable signal prior to beingcleaved and a second detectable signal after cleavage of the singlestranded detector DNA.

Aspect 145. The method according to any one of aspects 142-144, whereinthe fluorescence-emitting dye pair is a fluorescence resonance energytransfer (FRET) pair.

Aspect 146. The method according to aspect 142, wherein an amount ofdetectable signal increases after cleavage of the single strandeddetector DNA.

Aspect 147. The method according to any one of aspects 142-146, whereinthe fluorescence-emitting dye pair is a quencher/fluor pair.

Aspect 148. The method according to any one of aspects 142-147, whereinthe single stranded detector DNA comprises two or morefluorescence-emitting dye pairs.

Aspect 149. The method according to aspect 148, wherein said two or morefluorescence-emitting dye pairs include a fluorescence resonance energytransfer (FRET) pair and a quencher/fluor pair.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1

Metagenomic datasets from many diverse ecosystems were generated andhundreds of huge phage genomes, between 200 kbp and 716 kbp in length,were reconstructed. Thirty-four genomes were manually curated tocompletion, including the largest phage genomes yet reported. Expandedgenetic repertoires include diverse and new CRISPR-Cas systems, tRNAs,tRNA synthetases, tRNA modification enzymes, initiation and elongationfactors and ribosomal proteins. Phage CRISPR have the capacity tosilence host transcription factors and translational genes, potentiallyas part of a larger interaction network that intercepts translation toredirect biosynthesis to phage-encoded functions. Some phage repurposebacterial systems for phage-defense to eliminate competing phage. Sevenmajor clades of huge phage from human and other animal microbiomes,oceans, lakes, sediments, soils and the built environment werephylogenetically defined. It is concluded that large gene inventoriesreflect a conserved biological strategy, observed across a broadbacterial host range and resulting in the distribution of huge phageacross Earth's ecosystems.

Hundreds of phage sequences >200 kbp in length that were reconstructedfrom microbiome datasets generated from a wide variety of ecosystemswere presented. The three largest complete genomes for phage known todate, ranging up to 642 kbp in length, were reconstructed. A graphicalabstract provides an overview of the approach and main findings. Theresearch expands the understanding of phage biodiversity and brings tolight the variety of ecosystems in which phage have genome sizes thatrival those of small celled bacteria.

Ecosystem Sampling

Metagenomic datasets were acquired from human fecal and oral samples,fecal samples from other animals, freshwater lakes and rivers, marineecosystems, sediments, hot springs, soils, deep subsurface habitats andthe built environment (FIG. 5). For a subset of these, analyses ofbacterial, archaeal and eukaryotic organisms were published previously.Genome sequences that were clearly not bacterial, archaeal, archaealvirus, eukaryotic or eukaryotic virus were classified as either phage orplasmid-like based on their gene inventories. De novo assembledfragments of close to or >200 kbp in length were tested forcircularization and a subset selected for manual verification andcuration to completion (see Methods).

Genome Sizes and Basic Features

358 phage, 3 plasmid and 4 phage-plasmid sequences were reconstructed(FIG. 5).

Additional sequences inferred to be plasmids were excluded (seeMethods), and only those encoding CRISPR-Cas loci were retained (seebelow). Consistent with classification as phage, a wide variety ofphage-relevant genes were identified, including those involved in lysisand encoding structural proteins, and other expected phage genomicfeatures were documented.

Some phage predicted proteins are large, up to 7694 amino acids inlength. Many of these were tentatively annotated as structural proteins.180 phage sequences were circularized and 34 were manually curated tocompletion, in some cases by resolving complex repeat regions and theirencoded proteins (see Methods). Some genomes show a clear GC skew signalfor bi-directional replication, information that constrains theirreplication origin. The three largest complete, manually curated andcircularized phage genomes are 634, 636 and 643 kbp in length andrepresent the largest phage genomes reported to date. Previously, thelargest circularized phage genome was 596 kbp in length (Paez-Espino etal. (2016) supra). The same study reported a circularized genome of 630kbp in length, but this is an artifact. The problem of concatenatedsequences was sufficiently prominent in IMG-VR that these data were notincluded in further analyses. The complete and circularized genomes fromthe study, Refseq and published research were used to depict a currentview of the distribution of phage genome sizes (Methods). The mediangenome size for complete phage is ˜52 kbp (FIG. 1A), similar to theaverage size of ˜54 kbp reported previously (Paez-Espino et al. (2016)supra). Thus, sequences reported here substantially expand the inventoryof phage with unusually large genomes (FIG. 1B).

Intriguingly, two related sequences of 712 and >716 kbp in length wereidentified and manually curated (FIG. 5). These were classified as phagebased on their overall genome content and the presence of terminasegenes. The assemblies are confounded by few kb-long complex regionscomprised of small repeats at both genome ends. It is anticipated thatthese genomes could be closed if the repeat regions could berationalized.

Some genomes have very low coding density (nine <75%) due to use of agenetic code different from that used for gene prediction. A similarphenomenon was reported for Lak phage (Devoto et al. (2019) NatMicrobiol, and Ivanova et al. (2014) Science 344: 909-913). Distinctfrom prior studies, the genomes appear to use genetic code 16, in whichTAG, normally a stop codon, codes for an amino acid.

In only one case, a sequence of >200 kbp that was classified as aprophage based on transition into flanking bacterial genome sequence wasidentified. However, around half the genomes were not circularized, sotheir derivation from prophage cannot be ruled out. The presence ofintegrases in some genomes is suggestive of a lysogenic lifestyle undersome conditions.

Hosts, Diversity and Distribution

An intriguing question relates to the evolutionary history of phage withhuge genomes. Are they the result of recent genome expansion withinclades of normal sized phage or is a large inventory of genes anestablished, persistent strategy? To investigate this, phylogenetictrees for the large terminase subunit (FIG. 2) and major capsid proteinsusing as context sequences in public databases for phage of all sizeswere constructed (Methods). Many of the sequences from the large phagegenomes cluster together, defining clades. Analysis of the genome sizeinformation for database sequences shows that the public sequences thatfall into these clades are from phage with genomes of at least 120 kbpin length. The largest Glade, referred to here as Mahaphage (Maha beingSanskrit for huge), includes all of the present study's largest genomesas well as the Lak genomes from human and animal microbiomes (Devoto etal. (2019) supra). Six other clearly defined clusters of large phagewere identified, and they were named using the word for “huge” in avariety of languages. The existence of these clades establishes thatlarge genome size is a relatively stable trait. Within the seven clades,phage were sampled from a wide variety of environment types, indicatingdiversification of these large phage and their hosts across ecosystems.The environmental distribution of phage that are closely enough relatedthat their genomes largely can be aligned was also examined. In 17cases, these phage occur in at least two biotope types.

To determine the extent to which bacterial host phylogeny correlateswith phage clades, phage hosts were identified using CRISPR spacertargeting from bacteria in the same or related samples and phylogeny ofnormally host-associated genes that occur on phage (see below). Thepredictive value of bacterial affiliations of the phage gene inventorieswas also tested (Methods) and it was found that in every case, CRISPRspacer targeting and phylum-level phylogenetic profiling agreed withgene inventory characterizations. Consequently, this method was used topredict the phylum-level affiliations of hosts for many phage. Theresults establish the importance of firmicute and proteobacterial hosts,and indicate the higher prevalence of firmicute phage in the human andanimal gut compared to other environments (FIG. 5). Notably, the fourlargest genomes (634-716 kbp in length) are all for phage predicted toreplicate in Bacteroidetes, as do Lak phage with 540-552 kbp genomes(Devoto et al. (2019) supra), and all cluster within Mahaphage. Overall,phage grouped together phylogenetically are predicted to replicate inbacteria of the same phylum.

Metabolism, Transcription, Translation

The phage genomes encode proteins predicted to localize to the bacterialmembrane or cell surface. These may impact the susceptibility of thehost to infection by other phage. Almost all previously reportedcategories of genes suggested to augment host metabolism duringinfection were identified. Many phage have genes involved in steps of denovo biosynthesis of purines and pyrimidines and multiple steps thatinterconvert nucleic and ribonucleic acids and nucleotidephosphorylation states. These gene sets are intriguingly similar tothose of bacteria with very small cells and putative symbioticlifestyles (Castelle and Banfield (2018) Cell 172: 1181-1197).

Notably, many phage have genes whose predicted functions are intranscription and translation. Phage encode up to 64 tRNAs per genome,with sequences distinct from those of their hosts. Generally, the numberof tRNAs per genome increases with genome length (FIG. 1). They oftenhave up to 16 tRNA synthetases per genome, that are related to, butdistinct from, those of their hosts. Phage may use these proteins tocharge their own tRNA variants with host-derived amino acids. A subsetof genomes have genes for tRNA modification and to repair tRNAs cleavedas part of host defense against phage infection. Also identified are upto three probable ribosomal proteins per genome, the most common ofwhich is rpS21 (a phenomenon only recently reported in phage) (Mizuno etal. (2019) Nat. Commun. 10: 752); FIG. 3). Intriguingly, it is notedthat the phage rpS21 sequences have N-terminal extensions rich inarginine, lysine, and phenylalanine: residues that bind nucleic acids.It is predicted that these phage ribosomal proteins substitute for hostproteins in the ribosome (Mizuno et al. (2019) supra), and that theextensions protrude from the ribosome surface near the site oftranslation initiation to localize the phage mRNAs.

Some phage have genes predicted to function in other protein synthesissteps, including to ensure efficient translation. Several encode eitherinitiation factor 1 or 3 or both, sometimes as well as elongationfactors G, Tu, Ts and release factors. Also identified are genes thatencode ribosome recycling factors, along with tmRNAs and small protein B(SmpB) that rescue ribosomes stalled on damaged transcripts and triggerthe degradation of aberrant proteins. tmRNAs are also used by phages tosense the physiological state of host cells and can induce lysis whenthe number of stalled ribosomes in the host is high.

These observations suggest many ways in which some large phage cansubstantially intercept and redirect ribosome function. As phage mRNAsequences need to engage with the 3′ end of the host 16S rRNA toinitiate translation, their mRNA ribosomal binding sites were predicted.In the majority of cases, phage mRNAs have canonical Shine Dalgarno (SD)sequences, and an additional ˜15% have non-standard SD binding sites.Interestingly, however, phage whose genomes encode a probable orpossible rpS1 rarely have identifiable or canonical SD sequences. Thus,phage-encoded rpS1 may selectively initiate translation of phage mRNAs.Overall, phage genes appear to redirect the host's protein productioncapacity to favor phage genes by intercepting the earliest steps oftranslation. These inferences are aligned with findings for someeukaryotic viruses, which control every phase of protein synthesis(Jaafar and Kieft (2019) Nat. Rev. Microbiol. 17:110-123).Interestingly, some large putative plasmids also have analogous suitesof translation relevant genes.

About half of the phage genomes have one to fifty sequences >25 nt inlength that fold into perfect hairpins. The palindromes (sequences withdyad symmetry) are almost exclusively intergenic and each is uniquewithin a genome. Some, but not all, are predicted to be rho-independentterminators, thus provide clues regarding genes that function asindependently regulated units (Methods). However, some palindromes areup to 74 bp in length, and 34 genomes have examples of ≥40 nt in length,seemingly larger than normal terminators. These occur almost exclusivelyin Mahaphage and may have alternative or additional functions, such asmodulation of the movement of the mRNA through the ribosome.

CRISPR-Cas Mediated Interactions

Almost all major types of CRISPR-Cas systems on phage, including Cas9,the recently described Type V-I (Yan et al. (2019) Science 363: 88-91),and new subtypes of Type V-F systems were identified (Harrington et al.(2018) Science 362: 839-842.). The Class II systems (types II and V) arereported in phage for the first time. Most effector nucleases (forinterference) have conserved catalytic residues, implying that they maybe functional.

Unlike the previously well described case of a phage with a CRISPRsystem (Seed et al. (2013) Nature 494: 489-491), almost all phage CRISPRsystems lack spacer acquisition machinery (Cas1, Cas2, and Cas4) andmany lack recognizable genes for interference. For example, two relatedphage have both a Type I-C variant system lacking Cas1 and Cas2 and ahelicase protein in lieu of Cas3. They also harbor a second systemcontaining a new candidate 750 aa Type V effector protein that occursproximal to CRISPR arrays. In some cases, phage lacking genes forinterference and spacer integration have similar CRISPR repeats as theirhosts, thus may use Cas proteins synthesized by their host for thesefunctions. Alternatively the systems lacking an effector nuclease mayrepress transcription of the target sequences without cleavage (Luo etal. (2015) Nucleic Acids Res. 43:674-681; Stachler and Marchfelder(2016) J. Biol. Chem. 291:15226-15242).

The phage-encoded CRISPR arrays are often compact (3-55 repeats; median6 per array. This range is substantially smaller than typically found inbacterial genomes (Toms and Barrangou (2017) Biol. Direct 12:20). Somephage spacers target core structural and regulatory genes of otherphage. Thus, phage apparently augment their hosts' immune arsenal toprevent infection by competing phage.

Several large plasmid or plasmid-like genomes that encode a variety oftypes of CRISPR-Cas systems were identified. Some of these systems alsolack Cas1 and Cas2. Most commonly, the spacers target the mobilizationand conjugation-related genes of other plasmids, as well as nucleasesand structural proteins of phage.

Some phage-encoded CRISPR loci have spacers that target bacteria in thesame sample or in a sample from the same study. It is supposed that thetargeted bacteria are the hosts for these phage, an inference supportedby other host prediction analyses. Some loci with bacterialchromosome-targeting spacers encode Cas proteins that could cleave thehost chromosome, and some do not. Targeting of host genes could disableor alter their regulation, which may be advantageous during the phageinfection cycle. Some phage CRISPR spacers target bacterial intergenicregions, possibly interfering with genome regulation by blockingpromoters or silencing non-coding RNAs.

Among the most interesting examples of CRISPR targeting of bacterialchromosomes are genes involved in transcription and translation. Forinstance, one phage targets a σ⁷⁰ transcription factor in its host'sgenome, while encoding the gene for σ⁷⁰. There are previous reports ofσ⁷⁰ hijacking by phage with anti-sigma factors This may also occur withsome huge phage whose genomes encode anti-sigma factors. In anotherexample, a phage spacer targets the host Glycyl tRNA synthetase.

Interestingly, no evidence was found of targeting of any CRISPR-bearingphage by a host-encoded spacer, hinting at yet to be revealed componentsin phage-host-CRISPR interactions. However, phage CRISPR targeting ofother phage that are also targeted by bacterial CRISPR (FOG/4) suggestedphage-host associations that were broadly confirmed by the phagephylogenetic profile.

Some large Pseudomonas phage encode Anti-CRISPRs (Acr) (Bondy-Denomy etal. (2015) Nature 526:136-139; Pawluk et al. (2016) Nat Microbiol 1:16085) and proteins that assemble a nucleus-like compartment segregatingtheir replicating genomes from host defense and other bacterial systems.Proteins encoded in huge phage genomes that cluster with AcrVA5, AcrVA2,and AcrIIA7 that may function as Acrs were identified. Also identifiedwere tubulin-homologs (PhuZ) that position the “phage nucleus”, andproteins related to components of the proteinaceous barrier. Thus, phage‘nuclei’ may be a relatively common feature in large phage.

Methods

Phage and Plasmid Genome Identification

Datasets generated in the current study, those from prior research, theTara Oceans microbiomes (Karsenti et al. (2011) PLoS Biol. 9:e1001177),and the Global Oceans Virome (GOV; (Roux et al. (2016) Nature537:689-693) were searched for sequence assemblies that could havederived from phage with genomes of >200 kbp in length. Read assembly,gene prediction, and initial gene annotation followed standard methodsreported previously (Wrighton et al. (2014) ISME J. 8:1452-1463).

Phage candidates were initially found by retrieving sequences that werenot assigned to a genome and had no clear taxonomic profile at thedomain level. Taxonomic profiles were determined through a votingscheme, where there had to be a winner taxonomy >50% votes at eachtaxonomic rank based on Uniprot and ggKbase (ggkbase.berkeley.edu)database annotations. Phages were further narrowed down by identifyingsequences with a high number of hypothetical protein annotations and/orthe presence of phage structural genes, e.g. capsid, tail, holin. Allcandidate phage sequences were checked throughout to distinguishputative prophage from phage. Prophage were identified based on a cleartransition into genome with a high fraction of confident functionalpredictions, often associated with core metabolic functions, and muchhigher similarity to bacterial genomes. Plasmids were distinguished fromphage based on matches to plasmid marker genes (e.g. parA). Threesequence assemblies could not unambiguously be distinguished betweenphage and plasmid, and were assigned as “phage-plasmid”.

Phage and Plasmid Genome Manual Curation

All scaffolds classified as phage or phage-like were tested for endoverlaps using a custom script and checked manually for overlap.Assembled sequences that could be perfectly circularized were consideredpotentially “complete”. Erroneous concatenated sequence assemblies wereinitially flagged by searching for direct repeats >5 kb using Vmatch(Kurtz (2003) Ref Type: Computer Program 412:297). Potentiallyconcatenated sequence assemblies were manually checked for multiplelarge repeating sequences using the dotplot and RepeatFinder features inGeneious v9. Sequences were corrected and removed from further analysisif the corrected length was <200 kbp.

A subset of the phage sequences was selected for manual curation, withthe goal of finishing (replacing all N's at scaffolding gaps or localmisassemblies by the correct nucleotide sequences and circularization).Curation generally followed methods described previously (Devoto et al.(2019) supra). In brief, reads from the appropriate dataset were mappedusing Bowtie2 (Langmead and Salzberg (2012) Nat. Methods 9:357-359) tothe de novo assembled sequences. Unplaced mate pairs of mapped readswere retained with shrinksam (github.com/bcthomas/shrinksam). Mappingswere manually checked throughout to identify local misassemblies usingGeneious v9. N-filled gaps or misassembly corrections made use ofunplaced paired reads, in some cases using reads relocated from siteswhere they were mis-mapped. In such cases, mis-mappings were identifiedbased on much larger than expected paired read distances, highpolymorphism densities, backwards mapping of one read pair, or anycombination of the aforementioned.

Similarly, ends were extended using unplaced or incorrectly placedpaired reads until circularization could be established. In some cases,extended ends were used to recruit new scaffolds that were then added tothe assembly. The accuracy of all extensions and local assembly changeswere verified in a subsequent phase of read mapping. In many cases,assemblies were terminated or internally corrupted by the presence ofrepeated sequences. In these cases, blocks of repeated sequence as wellas unique flanking sequence were identified. Reads were then manuallyrelocated, respecting paired read placement rules and unique flankingsequences. After gap closure, circularization, and verification ofaccuracy throughout, end overlap was eliminated, genes were predictedand throughout, and the start moved to an intergenic region, in somecases suspected to be origin based on a combination of coverage trendsand GC skew (Brown et al. (2016) Nat. Biotechnol. 34:1256-1263).Finally, the sequences were checked to identify any repeated sequencesthat could have led to an incorrect path choice because the repeatedregions were larger than the distance spanned by paired reads. This stepalso ruled out artifactual long phage sequences generated by end to endrepeats of smaller phage, which occur in previously described datasets.

Structural and Functional Annotation

Following identification and curation of phage genomes, coding sequences(CDS) were predicted with prodigal (-m -c -g 11 -p single) with geneticcode 11. The resulting CDS were annotated as previously described bysearching against UniProt, UniRef, and KEGG (Wrighton et al. (2014)supra). Functional annotations were further assigned by searchingproteins against Pfam r32 (Finn et al. (2014) Nucleic Acids Res.42:D222-30), TIGRFAMS r15 (Haft et al. (2013) Nucleic Acids Res.41:D387-95), and Virus Orthologous Groups r90 (vogdb.org). tRNAs wereidentified with tRNAscan-SE 2.0 (Lowe and Eddy, (1997) Nucleic AcidsRes. 25: 955-964) using the bacterial model. tmRNAs were assigned usingARAGORN v1.2.38 (Laslett and Canback, (2004) Nucleic Acids Res. 32:11-16) with the bacterial/plant genetic code. Clustering of the proteinsequences into families was achieved using a two-step procedure. A firstprotein clustering was done using the fast and sensitive proteinsequence searching software MMseqs (Hauser et al. (2016) Bioinformatics32: 1323-1330). An all-vs-all sequences search was performed usinge-value: 0.001, sensitivity: 7.5 and coverage: 0.5. A sequencesimilarity network was built based on the pairwise similarities and thegreedy set cover algorithm from MMseqs was performed to define proteinsubclusters. The resulting subclusters were defined as subfamilies. Inorder to test for distant homology, subfamilies were grouped intoprotein families using an HMM-HMM comparison. The proteins of eachsubfamily with at least two protein members were aligned using theresult2msa parameter of mmseqs2, and from the multiple sequencealignments HMM profiles were built using the HHpred suite. Thesubfamilies were then compared to each other using HHblits (Remmert etal. (2011) Nat. Methods 9: 173-175 from the HHpred suite (withparameters -v 0 -p 50 -z 4 -Z 32000 -B 0 -b 0). For subfamilies withprobability scores of ≥95% and coverage ≥0.50, a similarity score(probability×coverage) was used as weights of the input network in thefinal clustering using the Markov Clustering algorithm, with 2.0 as theinflation parameter. These clusters were defined as the protein familiesHairpins (palindromes, based on identical overlapping repeats in theforward and reverse directions) were identified using the GeneiousRepeat Finder and located dataset-wide using Vmatch (Kurtz (2003)supra). Repeats >25 bp with 100% similarity were tabulated.

Reference Genomes for Size Comparisons

RefSeq v92 genomes were recovered by using the NCBI Virus portal andselecting only complete dsDNA genomes with bacterial hosts. Genomes from(Paez-Espino et al. (2016) supra) were downloaded from IMG/VR and onlysequence assemblies labeled “circular” with predicted bacterial hostswere retained. Many of the genomes were the result of erroneousconcatenated repeating assemblies. Given the presence of sequences inIMG/VR that are based on erroneous concatenations, the study onlyconsidered sequences from this source that are >200 kb; a subset ofthese were removed as artifactual sequences.

Host Prediction

The phylum affiliations of bacterial hosts for phage were predicted byconsidering the Uniprot taxonomic profiles of every CDS for each phagegenome. The phylum level matches for each phage genome were summed andthe phylum with the most hits was considered as the potential hostphylum. However, only cases where this phylum that had 3× as many countsas the next most counted phylum were assigned as the tentative phagehost phylum. Phage hosts were further assigned and verified using CRISPRtargeting. CRISPR arrays were predicted on sequence assemblies >1 kbpfrom the same environment that each phage genome was reconstructed.Spacers were extracted and searched against the genomes from the samesite using BLASTN-short (Altschul et al. (1990) J. Mol. Biol.215:403-410). Sequence assemblies containing spacers with a match oflength >24 bp and ≤1 mismatch or at least 90% sequence identity to agenome were considered targets. In the case of phage, the match was usedto infer a phage-host relationship. In all cases, the predicted hostphylum based on taxonomic profiling and CRISPR targeting were incomplete agreement. Similarly, the phyla of hosts were predicted basedon phylogenetic analysis of phage genes also found in host genomes(e.g., involved in translation and nucleotide reactions). Inferencesbased on computed taxonomic profiles and phylogenetic trees were also incomplete agreement.

Alternative Genetic Codes

In cases where gene prediction using the standard bacterial code (code11) resulted in seemingly anomalously low coding densities, potentialalternative genetic codes were investigated. In addition to making aprediction using Fast and Accurate genetic Code Inference and Logo(FACIL; (Dutilh et al. (2011) Bioinformatics 27:1929-1933)), genes withwell defined functions (e.g., polymerase, nuclease) were identified andthe stop codons terminating genes that were shorter than expected weredetermined. Genes were then re-predicted using Glimmer and Prodigal setsuch that codon was not interpreted as a stop. Other combinations ofrepurposed stop codons were evaluated, and candidate codes (e.g., code6, with only one stop codon) were ruled out due to unlikely gene fusionpredictions.

Introns were identified in some longer than expected pseudo-tRNAs byre-predicting the tRNAs using eukaryotic settings (as tRNA scan does notexpect introns in tRNA genes in bacteria and phage).

Terminase Phylogenetic Analysis

The large terminase phylogenetic tree was constructed by recoveringlarge terminases from the aforementioned annotation pipeline. CDS thatmatched with >30 bitscore against PFAM, TIGRFAMS, and VOG were retained.Any CDS that had a hit to large terminase, regardless of bitscore, wassearched using HHblits (Steinegger et al. Bioinformatics 21:951-960)against the uniclust30_2018_08 database. The resulting alignment wasthen further searched against the PDB70 database. Remaining CDS thatclustered in protein families with a large terminase HMM were alsoincluded after manual verification. Detected large terminases weremanually verified using HHPred (Steinegger et al. supra) and jPred (Coleet al. (2008) Nucleic Acids Res. 36:W197-201). Large terminases fromthe >200 kb (Paez-Espino et al. (2016) supra) phage genomes and all >200kb complete dsDNA phage genomes from RefSeq r92 were also included byprotein family clustering with the phage CDS from this study. Theresulting terminases were clustered at 95% amino acid identity (AAI) toreduce redundancy using cd-hit (Huang et al. (2010) Bioinformatics26:680-682) Smaller phage genomes were included by searching theresulting CDS set against the Refseq protein database and retaining thetop 10 best hits. Those hits that had no large terminase match againstPFAM, TIGRFAMS, or VOG were removed from further consideration and theremaining set was clustered 90% AAI. The final set of large terminaseCDS were aligned MAFFT v7.407 (--localpair --maxiterate 1000) and poorlyaligned sequences were removed and the resulting set was realigned. Thephylogenetic tree was inferred using IQTREE v1.6.9 (Nguyen et al. (2015)Mol. Biol. Evol. 32:268-274).

Phage Encoded tRNA Synthetase Trees

Phylogenetic trees were constructed for phage encoded tRNA synthetase,ribosomal and initiation factor protein sequences using a set of theclosest set of reference from NCBI and bacterial genomes from thecurrent study.

CRISPR-Cas Locus Detection and Host Identification

Phage-encoded CRISPR-Cas loci were identified using the same methods asused to identify bacterial CRISPR-Cas loci, spacers extracted frombetween repeats of the CRISPR locus using MinCED(github.com/ctSkennerton/minced) and CRISPRDetect (Biswas et al., 2016)were compared to sequences reconstructed from the same site and targetsclassified as bacterial, phage or other.

Because many phage hosts cannot be identified by CRISPR targeting(perhaps because phage had proliferated in samples containing sensitivehosts, or the targets are sufficiently mutated to avoid spacerdetection) additional lines of evidence were used to propose hostidentities. Due to uncertainty in these methods, possible phagepredictions were made only at the phylum level. In this analysis, thefraction of genes encoded on any genome with the best predicted proteinmatch to each phylum was computed. Only in cases where the most highlyrepresented phylum exceeded in frequency the next most common phylum by≥3× was a tentative bacterial host proposed. This threshold was verifiedas conservative, based on confirmed host phylum information from CRISPRtargeting or phylogenetic analysis.

Data Availability

Supplementary document “Genbank” includes the Genbank format files forthe genome sequences reported in this study. All reads are beingdeposited in the short read archive (if not already lodged there) andgenome sequences in NCBI.

Example 2

Cas12J represents the smallest known single-effector Cas protein withdouble-stranded DNA (dsDNA) targeting ability. Cas12J is capable ofcleaving dsDNA without a requirement for an accessory RNA (e.g. such asa tracrRNA) to function. Additionally, the RuvC domain, which is the ahighly conserved domain across Cas12 and Cas9, is highly divergent inCas12J from known Cas proteins, and the domain architecture is differentacross members of the Cas12 protein superfamily.

Results

To investigate the functionality and DNA targeting capability of theCas12J effector in a heterologous context, an efficiency oftransformation (EOT) plasmid interference assay was set up (FIG. 11A).Escherichia coli BL21(DE3) expressing cas12J and a crRNA guide targetingthe antisense strand of the bla gene, or a non-targeting guide, weretransformed with pUC19 (FIG. 11B). The assay revealed that the pUC19transformation efficiency is reduced by 2-3 orders of magnitude instrains producing Cas12J and the pUC19 targeting guide, compared tostrains producing Cas12J and the non-targeting guide (FIG. 11C). Thisresult is indicative of a robust and guide dependent double-stranded DNAinterference activity of Cas12J. To assess the DNA interference unbiasedrelative transformation efficiency of each strain, the pYTK001 plasmidwas transformed as a control (FIG. 11B). The transformation efficiencyrevealed that the strains are equally competent for transformation of anon-targeted plasmid (FIG. 11C).

Methods

Cloning of the Expression Plasmids

The gene sequence of cas12J from contigP0_An_GD2017L_S7_coassembly_k141_3339380 was ordered as a G-block fromIDT and cloned into pRSFDuet-1 (Novagen) into MCSI using Golden Gateassembly. In the same reaction a T7 promotor, the respective consensusrepeat sequence from the CRISPR-array located on contigP0_An_GD2017L_S7_coassembly_k141_3339380, together with a 35 bp spaceramenable to Golden Gate assembly mediated spacer exchange wereintroduced downstream of the cas12J ORF in place of MCSII. In the samereaction a hepatitis delta virus ribozyme (HDVrz) was introduceddownstream of the spacer to facilitate homogeneous processing of theimmature crRNA transcript at its 3′-terminus. To generate the pUC19targeting Cas12J-vector, the non-targeting spacer was exchanged byGolden Gate assembly to a sequence matching base pairs 11-45 of thepUC19 bla gene downstream of the AGTATTC sequence, to allow forproduction of an antisense strand complementary crRNA guide.

Plasmid Interference Assay

The generated Cas12J vectors (non-targeting and pUC19-targeting) weretransformed in chemically competent E. coli BL21(DE3) (NEB). Threeindividual colonies for each strain (A, B and C strains) were picked toinoculate three 5 mL (LB, Kanamycin 50 μg/mL) starter cultures toprepare electrocompetent cells the following day. 50 mL (LB, Kanamycin50 μg/mL) main cultures were inoculated 1:100 and grown vigorouslyshaking at 37° C. to an OD₆₀₀ of 0.3. Subsequently, the cultures werecooled to room temperature and cas12J expression was induced with 0.2 mMIPTG. Cultures were grown to an OD₆₀₀ of 0.6-0.7 at 25° C. for 1 h,before preparation of electrocompetent cells by repeated ice-cold ddH₂Oand 10% glycerol washes. Cells were resuspended in 250 μL 10% glycerol.90 μL aliquots were flash frozen in liquid nitrogen and stored at −80°C. The next day, 80 μL competent cells were combined with 3.2 μL plasmid(20 ng/μL pUC19 target plasmid, or 20 ng/μL pYTK001 control plasmid),incubated for 30 min on ice and split into three individual 25 μLtransformation reactions. After electroporation in 0.1 mmelectroporation cuvettes (Bio-Rad) on a Micropulser electroporator(Bio-Rad), cells were recovered in 1 mL recovery medium (Lucigen)supplemented with 0.2 mM IPTG, shaking at 37° C. for one hour.Subsequently, 10-fold dilution series were prepared and 5 μL of therespective dilution steps were spot-plated on LB-Agar containing theappropriate antibiotics. Plates were incubated over night at 37° C. andcolonies were counted the following day to determine the transformationefficiency. To assess the transformation efficiency, the mean andstandard deviations were calculated from the cell forming units per ngtransformed plasmids for the electroporation triplicates.

FIG. 11A-11C shows the efficiency of transformation plasmid interferenceassay. FIG. 11A upper panel: experimental scheme. E. coli producingCas12J are transformed with a targeted plasmid (pUC19). Lower panel:vector map of the effector expression plasmid. FIG. 11B, serialdilutions of E. coli producing Cas12J and either pUC19-targeting ornon-targeting guides, transformed with pUC19 (left) or pYTK001 (right).FIG. 11C, calculated transformation efficiencies in cell forming units(cfu) per ng transformed plasmid. Mean and +/−s.d. (error bars) valueswere derived from triplicates.

Example 3

Results

To demonstrate that Cas12J cuts dsDNA—in vitro experiments outside ofcells (i.e., in a non-cellular context) were performed. Linear dsDNA wascleaved in the presence of Cas12J and a guide RNA designed to hybridizeto a target sequence adjacent to a PAM motif. The Cas12Jribonucleoprotein (RNP) complex was either assembled inside of cells (E.coli in this case via the introduction of plasmid DNA encoding theprotein and the guide RNA), or assembled in vitro outside of cells fromapo protein and synthetic RNA oligonucleotides. The experiment revealedthat RNPs with Cas12J-1947455 (“Ortholog #1”), Cas12J-2071242 (“Ortholog#2”), or Cas12J-3339380 (“Ortholog #3”) assembled either inside oroutside of cells cleaved linear dsDNA fragments guided by the crRNAspacer sequence of the guide RNA (FIG. 12A and FIG. 12B). The 1.9 kblinear DNA substrate was cleaved into 1.2 kb and a 0.7 kb fragment,indicative of an endonucleolytic DNA double strand cleavage event closeto the site of guide complementarity. dsDNA cleavage was not observed inthe absence of a guide complementary site on the DNA. This experimentdemonstrated that Cas12J (e.g., Cas12J-1947455, Cas12J-2071242 andCas12J-3339380) is a crRNA guided DNA-endonucleases capable ofintroducing double strand breaks into DNA. Furthermore, the experimentdemonstrated that functional Cas12J RNPs can be assembled inside and/oroutside of cells.

FIG. 12A-12B demonstrates that Cas12J (e.g., Cas12J-1947455,Cas12J-2071242 and Cas12J-3339380) cleave linear dsDNA fragments guidedby a crRNA spacer sequence. FIG. 12A, Time dependent dsDNA cleavageassays for the RNPs that were assembled inside of cells. top:Cas12J-1947455 (Cas12J-1), middle: Cas12J-2071242 (Cas12J-2) and bottom:Cas12J-3339380 (Cas12J-3). The far right lanes are non-complementary DNAcontrols, which could not be identified by the respective crRNA guide.FIG. 12B, Time dependent dsDNA cleavage assays for the RNPs that wereassembled in vitro outside of cells. top: Cas12J-1947455 (Cas12J-1),middle: Cas12J-2071242 (Cas12J-2) and bottom: Cas12J-3339380 (Cas12J-3).The far right lanes are non-complementary DNA controls, which could notbe identified by the respective crRNA guide.

PAM depletion assays were performed in Escherichia coli. In the assay,Cas12J targets a DNA sequence adjacent to a randomized sequence in aplasmid library. NGS sequencing revealed that Cas12J and crRNA weresufficient in bacteria to deplete plasmids with crRNA guidecomplementary target DNA sites, when a T-rich PAM sequence was adjacentto the protospacer (FIG. 13). The experiment also showed that notracrRNA was required for the formation of functional effectors.Noteworthy, ortholog #2 features a minimal 5′-TBN-3′ PAM sequence.

FIG. 13. PAM sequences depleted by the three different orthologs,demonstrating that PAMs are straightforward to identify for any desiredCas12J protein.

Methods

Cloning of the Expression Constructs

The gene sequences of Cas12J-1947455, Cas12J-2071242 and Cas12J-3339380were ordered as G-blocks from IDT and cloned into pRSFDuet-1 (Novagen)into MCSI C-terminally fused to a hexa-histidine tags using Golden Gateassembly. For co-expression of cas12J with crRNA guides, CRISPR-arrays(36 bp repeat followed by a 35 bp spacer, six units thereof) were clonedunder the control of a T7-promoter in high copy vectors (ColE1 origin),which contained bla genes for selection.

Production of the Cas12J-RNP In Vivo and Purification

The generated cas12J overexpression vectors and CRISPR array expressionvectors were co-transformed in E. coli BLR(DE3) (Novagen) and incubatedover night at 37° C. on LB-Kan-Carb agar plates (50 μg/mL Kanamycin, 50μg/mL Carbenicillin).). Single colonies were picked to inoculate 80 mL(LB, Carbenicillin 50 μg/mL and Kanamycin 50 μg/mL) starter cultureswhich were incubated at 37° C. shaking vigorously overnight. The nextday, 1.5 L TB-Kan-Carb medium (Carbenicillin 50 μg/mL and Kanamycin 50μg/mL) were inoculated with the respective 40 mL starter culture andgrown at 37° C. to an OD₆₀₀ of 0.6, cooled down on ice for 15 min andgene expression was subsequently induced with 0.5 mM IPTG followed byincubation over night at 16° C. Cells were harvested by centrifugationand resuspended in wash buffer (50 mM HEPES-Na (pH 7.5), 500 mM NaCl, 20mM imidazole, 5% glycerol and 0.5 mM TCEP), subsequently lysed bysonication followed by lysate clarification by centrifugation. Thesoluble fraction was loaded on a 5 mL Ni-NTA Superflow Cartridge(Qiagen) pre-equilibrated in wash buffer. Bound proteins were washedwith 20 column volumes (CV) wash buffer and subsequently eluted in 3 CVelution buffer (50 mM HEPES-Na (pH 7.5), 500 mM NaCl, 500 mM imidazole,5% glycerol and 0.5 mM TCEP). Eluted proteins were dialyzed over nightat 4° C. in slide-a-lyzer dialysis cassettes 10 k mwco (Thermo FisherScientific) against ion-exchange (IEX) loading buffer (20 mM Tris pH9.0, 4° C., 125 mM NaCl, 5% glycerol and 0.5 mM TCEP). Proteins wereloaded onto 2×5 mL HiTrap Q HP anion exchange chromatography columns.Proteins were eluted in a gradient of IEX elution buffer (20 mM Tris pH9.0, 4° C., 1 M NaCl, 5% glycerol and 0.5 mM TCEP). Elution fractionswere analyzed by SDS-PAGE and Urea-PAGE and fraction containing RNPformed by Cas12J and crRNA were concentrated to 1 mL. Finally, proteinswere injection into a HiLoad 16/600 Superdex® 200 pg columnpre-equilibrated in size-exclusion buffer (10 mM HEPES-Na (pH 7.5), 150mM NaCl and 0.5 mM TCEP). Peak fractions were concentrated to anabsorption at 280 nm of 60 AU (NanoDrop® 8000 Spectrophotometer, ThermoScientific), corresponding to an estimated concentration of 500 μM.Subsequently, proteins were snap frozen in liquid nitrogen and stored at−80° C.

Production and Purification of Apo Cas12J

The generated cas12J overexpression vectors were transformed inchemically competent E. coli BL21(DE3) (NEB) and incubated over night at37° C. on LB-Kan agar plates (50 μg/mL Kanamycin). Single colonies werepicked to inoculate 80 mL (LB, Kanamycin 50 μg/mL) starter cultureswhich were incubated at 37° C. shaking vigorously overnight. The nextday, 1.5 L TB-Kan medium (50 μg/mL Kanamycin) were inoculated with therespective 40 mL starter culture and grown at 37° C. to an OD₆₀₀ of 0.6,cooled down on ice for 15 min and gene expression was subsequentlyinduced with 0.5 mM IPTG followed by incubation over night at 16° C.Cells were harvested by centrifugation and resuspended in wash buffer(50 mM HEPES-Na (pH 7.5), 1 M NaCl, 20 mM imidazole, 5% glycerol and 0.5mM TCEP), subsequently lysed by sonication followed by lysateclarification by centrifugation. The soluble fraction was loaded on a 5mL Ni-NTA Superflow Cartridge (Qiagen) pre-equilibrated in wash buffer.Bound proteins were washed with 20 column volumes (CV) wash buffer andsubsequently eluted in 5 CV elution buffer (50 mM HEPES-Na (pH 7.5), 500mM NaCl, 500 mM imidazole, 5% glycerol and 0.5 mM TCEP). The elutedproteins were concentrated to 1 mL before injection into a HiLoad 16/600Superdex® 200 pg column pre-equilibrated in size-exclusion buffer (20 mMHEPES-Na (pH 7.5), 500 mM NaCl, 5% glycerol and 0.5 mM TCEP). Peakfractions were concentrated to an absorption at 280 nm of 40 AU(NanoDrop® 8000 Spectrophotometer, Thermo Scientific), corresponding toan estimated concentration of 500 μM. Subsequently, proteins were snapfrozen in liquid nitrogen and stored at −80° C.

Cas12J-crRNA RNP Reconstitution

Cas12J-crRNA RNP complexes were assembled at a concentration of 1.25 μMby mixing protein and synthetic crRNA (IDT) in a 1:1 molar ratio inreconstitution buffer (10 mM Hepes-K pH 7.5, 150 mM KCl, 5 mM MgCl₂, 0.5mM TCEP) and incubation at 20° C. for 30 min. The synthetic crRNA wasprior to the assembly reaction heated to 95° C. for 3 min and thencooled down to RT for proper folding.

DNA Cleavage Assay

DNA target substrates were generated by PCR from plasmid template DNA.Cleavage reactions were initiated by addition of DNA (10 nM) topreformed RNP (1 μM) in reaction buffer (10 mM Hepes-K pH 7.5, 150 mMKCl, 5 mM MgCl₂, 0.5 mM TCEP). The reactions were incubated at 37° C.and aliquots were removed at the indicated intervals, quenched with 50mM EDTA and stored in liquid nitrogen. After completion of thetime-series, samples were thawed and treated with 0.8 units proteinase K(NEB) for 20 min at 37° C. Loading dye was added (Gel Loading Dye Purple6X, NEB) and samples were analyzed by electrophoresis on an 1% agarosegel.

Sequences Used

crRNA guides: >crRNA-1 (guide sequence/targeting sequence is in bold)(SEQ ID NO: 99) CACAGGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGACAGCUGGUAAUGGGAUACCUU >crRNA-2 (guide sequence/targeting sequence is in bold)(SEQ ID NO: 100) UAAUGUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGACUGCCGCCUCCGCGACGCCCA >crRNA-3 (guide sequence/targeting sequence is in bold)(SEQ ID NO: 101) AUUAACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGACUAUGAGCUUAUGUACAUCAA

DNA targets (PAM motifs are underlined crRNA spacer complementarysequences are bold):

>Linear pTarget1: (SEQ ID NO: 102)gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgacttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggccgccccttgtaGTTAagctggtaatgggataccttAtacagcggccgcgattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcg >linear pTarget2: (SEQ ID NO: 103)gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgacttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggccgccccttgtatTTCTGCCGCCTCCGCGACGCCCAatacagcggccgcgattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcg >linear pTarget3:(SEQ ID NO: 104)gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgacttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggccgccccttgtaATTCtatgagCttatgtacatcaaAtacagcggccgcgattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcg

Example 4

Results

Transcriptomic mapping suggested that crRNA was expressed heterologouslyin E. coli cells and processed to include a 25 nucleotide-long repeatand a 14-20 nucleotide spacer. The data also suggested that Cas12Jlikely processes its own crRNA (see FIG. 14A-14C).

FIG. 14A-14C illustrates results from mapping RNA sequences to theCas12J CRISPR locus from pBAS::Cas12J-1947455 (FIG. 14A),pBAS::Cas12J-2071242 (FIG. 14B), and pBAS::Cas12J-3339380 (FIG. 14C).Inset shows a detailed view of transcriptome mapping to the firstrepeat-spacer-repeat iteration in each locus. Black diamonds denoterepeats; squares denote spacers; faded repeats and spacers denote thedegenerate end of the array.

Methods

RNA-Seq

pBAS::Cas12J-1947455, pBAS::Cas12J-2071242, and pBAS::Cas12J-3339380constructs were transformed in chemically competent E. coli DH5α(QB3-Macrolab, UC Berkeley) and incubated over night at 37° C. on LB-Cmagar plates (34 μg/mL chloramphenicol). Single colonies were picked toinoculate 5 mL (LB, 34 μg/mL chloramphenicol) starter cultures whichwere incubated at 37° C. shaking vigorously overnight. The next morning,main cultures were inoculated 1:100 (LB, 34 μg/mL chloramphenicol) andlocus expression was induced with 200 nM aTc for 24 h at 16° C. Cellswere harvested by centrifugation, resuspended in lysis buffer (20 mMHepes-Na pH 7.5, 200 mM NaCl) and lysed using glass beads (0.1 mm glassbeads, 4×30 s vortex at 4° C., interspaced by 30 s cool-down on ice).200 μL cell lysis supernatant were transferred into Trizol® for RNAextraction according to the manufacturers protocol (Ambion). 10 μg RNAwere treated with 20 units of T4-PNK (NEB) for 6 h at 37° C. fordephosphorylation. Subsequently, 1 mM ATP was added and the sample wasincubated for 1 h at 37° C. for 5′-phosphorylation before heatinactivation at 65° C. and subsequent Trizol® purification.

Next, cDNA libraries were prepared using the RealSeq-AC miRNA librarykit illumina sequencing (somagenics). cDNA libraries were subjected toIllumina MiSeq® sequencing, generating 50 nucleotide-long single reads.Raw sequencing data was processed to remove adapters and sequencingartifacts, and high-quality reads were maintained. The resulting readswere mapped to their respective plasmids to determine the CRISPR locusexpression and crRNA processing.

Example 5

Results

The data provided in FIG. 15 show that Cas12J can induce targeted GFPdisruption, indicating successful Non-Homologous End Joining (NHEJ) andtargeted genomic editing in human cells. In one case, an individualCas12J/guide RNA was able to edit as high as 33% of cells (Cas12J-2guide 2), comparable to levels reported for CRISPR-Cas9, CRISPR-Cas12a,and CRISPR-CasX (Coag et al. (2013) Science 339:819; Jinek et al (2013)eLife, 2:e00471; Mali et al. (2013) Science 339:823; and Liu et al.(2019) Nature 566:7743).

Methods

Cloning of Cas12J Effector Plasmids for Expression in Human Cell

The gene sequence of cas12J-2 and cas12J-3 were ordered as G-blocks fromIntegrated DNA Technologies (IDT) encoding codon optimized genes forexpression in human cells. G-blocks were cloned via Golden Gate assemblyinto the vector backbone of pBLO62.5, downstream fused to two SV40 NLSsvia a GSG linker encoding sequence (FIG. 16A-16B, providing constructmaps; and Table 1 (provided in FIG. 17A-17G), providing nucleotidesequences of the constructs). The guide encoding sequence of pBLO62.5was exchanged to encode for a single CRISPR-repeat of the respectivehomologue, followed by a 20 bp stuffer spacer sequence amenable toGolden Gate exchange using the restriction enzyme SapI (FIG. 16A-16B;and Table 1 (provided in FIG. 17A-17G)). To generate EGFP targetingconstructs, the stuffer was exchanged via Golden Gate assembly to encodethe guide for the selected target site (Table 2).

TABLE 2 Guide sequences Guide # Spacer Sequence 5′->3′ NTCGTGATGGTCTCGATTGAGT (SEQ ID NO: 105) 1ACCGGGGTGGTGCCCATCCT (SEQ ID NO: 106) 2ATCTGCACCACCGGCAAGCT (SEQ ID NO: 107) 3GAGGGCGACACCCTGGTGAA (SEQ ID NO: 108)Human-Cell Targeted GFP Disruption

The GFP HEK293 reporter cells were previously generated via lentiviralintegration as previously described. Antony et al. (2018) Mol. Cell.Pediatrics 5:9. Cells were routinely tested for Mycoplasma using theMycoAlert Mycoplasma Detection Kit (Lonza), according to themanufacturer's protocol. GFP HEK293 reporter cells were seeded into96-well plates and transfected the next day with Lipofectamine® 3000(Life Technologies) and 200 ng of plasmid DNA encoding the Cas12J gRNAand Cas12J-P2A-puromycin fusion. 24 hours post-transfection,successfully transfected cells were selected for by adding 1.5 puromycinto the cell culture media for 72 hours. Cells were passaged to maintainsub-confluent conditions and then analyzed on an Attune NxT HowCytometer with an autosampler. Cells were analyzed on the flow cytometerafter 7 days to allow for clearance of GFP from cells.

Example 6

Results

To test whether Cas12J features an unspecific trans-cleavage activity,once activated by cis-targeted nucleic acids, an in vitro cleavage assaywas set up. In the assay, the Cas12J RNPs and trans cleavage ssDNA orssRNA substrates were incubated in the presence of no cis-activator,ssDNA cis-activator, dsDNA cis-activator, or ssRNA cis-activator.

As shown in FIG. 18, the three tested Cas12J homologs efficiently cleavessDNA, but not ssRNA, when an activating DNA, but not RNA, is present inthe reaction. This assay demonstrates that Cas12J can be activated byspacer complementary ssDNA, or dsDNA, to target ssDNA in trans.Furthermore, this DNA-activated ssDNA trans cleavage activity can beused for nucleic acid detection using a Fluorophore-quencher labeledreporter assay (East-Seletsky et al., Nature 538, 270-273 (2016)).

Methods

ssDNA and ssRNA substrates for trans cleavage were designed to benon-complementary to the spacer of the Cas12J guide RNA. Substrates were5′-end-labelled using T4-PNK (NEB) in the presence of ³²P-γ-ATP. ActiveCas12J RNP complexes were assembled by diluting Cas12J protein and guidecrRNA to 4 μM in complex assembly buffer (20 mM HEPES-Na pH 7.5 RT, 300mM KCl, 10 mM MgCl₂, 20% glycerol, 1 mM TCEP) and incubation for 30 minat RT. Spacer complementary activator substrates were diluted inoligonucleotide hybridization buffer (10 mM Tris pH 7.8 RT, 150 mM KCl)to a concentration of 4 μM, heated to 95° C. for 5 min, and subsequentlycooled down at room temperature (RT) to allow duplex formation fordouble stranded activator substrates. Cleavage reactions were set up bycombining 200 nM RNP with 400 nM activator substrate and incubation for10 min at RT before addition of 2 nM ssDNA, or ssRNA, trans cleavagesubstrates. Reactions were conducted in reaction buffer (10 mM HEPES-NapH 7.5 RT, 150 mM KCl, 5 mM MgCl₂, 10% glycerol, 0.5 mM TCEP) andincubated for 60 min at 37° C. Reactions were stopped by addition of twovolumes formamide loading buffer (96% formamide, 100 μg/mL bromophenolblue, 50 μg/mL xylene cyanol, 10 mM EDTA, 50 μg/mL heparin), heated to95° C. for 5 min, and cooled down on ice before separation on a 12.5%denaturing urea-polyacrylamide gel electrophoresis (PAGE). Gels weredried for 4 h at 80° C. before phosphor-imaging visualization using anAmersham Typhoon™ scanner (GE Healthcare).

Example 7

Materials and Methods

Metagenomic Assemblies, Genome Curation, and CRISPR-CasΦ (CRISPR-Cas12J)Detection

Metagenomic sequencing data was assembled using previously describedmethods (Peng et al. Bioinformatics. 28, 1420-1428 (2012); and Nurk etal. Genome Res. 27, 824-834 (2017). Coding sequences (CDS) werepredicted from sequence assemblies using prodigal with genetic code 11(-m -g 11 -p single) and (-m -g 11 -p meta) and preliminary annotationswere performed as previously described by searching against UniProt,UniRef100, and KEGG (Wrighton et al. ISME J. 8, 1452-1463 (2014)). Phagegenome curation was performed as described above. Briefly, Bowtie2v2.3.4.1 (Langmead and Salzberg Nat. Methods. 9, 357-359 (2012)) wasused to map reads to the de novo assembled sequences, and unplaced matepairs of mapped reads were retained with shrinksam(github.com/bcthomas/shrinksam). N-filled gaps and local misassemblieswere identified and corrected, and unplaced or incorrectly placed pairedreads allowed extension of contig ends. Local assembly changes andextensions were verified with further read mapping. A database of CasΦsequences was generated using MAFFT v7.407 (Katoh and Standley Mol.Biol. Evol. 30, 772-780 (2013)) and hmmbuild. CDS from new assemblieswere searched against the HMM database using hmmsearch with e-value<1×10⁻⁵ and added to the database upon verification.

Phylogenetic Analysis of Type V Systems

Cas protein sequences were collected as described above andrepresentatives from the TnpB superfamily were collected from Makarovaet al. (Nat. Rev. Microbiol., 1-17 (2019)) and top BLAST hits fromRefSeq. The resulting set was clustered at 90% amino acid identity usingCD-HIT to reduce redundancy (Huang et al. Bioinformatics. 26, 680-682(2010)). A new alignment of CasΦ with the resulting sequence set wasgenerated using MAFFT LINSI with 1000 iterations and filtered to removecolumns comprised of gaps in 95% of sequences. Poorly aligned sequenceswere removed and the resulting set was realigned. The phylogenetic treewas inferred using IQTREE v1.6.6 using automatic model selection (Nguyenet al. Mol. Biol. Evol. 32, 268-274 (2015)) and 1000 bootstraps.

crRNA Sequence Analysis

CRISPR-RNA (crRNA) repeats from Phage-encoded CRISPR loci wereidentified using MinCED (github.com/ctSkennerton/minced) andCRISPRDetect (Biswas et al. BMC Genomics. 17, 356 (2016)). The repeatswere compared by generating pairwise similarity scores using theNeedleman-Wunsch algorithm followed by EMBOSS Needle (McWilliam et al.Nucleic Acids Res. 41, W597-600 (2013)). A heatmap was built using thesimilarity score matrix and hierarchical clustering produced dendrogramsthat were overlaid onto the heatmap to delineate different clusters ofrepeats.

Generation of Plasmids

CasΦ loci, including an additional E. coli RBS upstream of casΦ, wereordered as G-blocks from Integrated DNA Technologies (IDT) and clonedusing Golden Gate assembly (GG) under the control of atetracycline-inducible promoter for RNA seq and PAM depletion plasmidinterference experiments. Perfect repeat-spacer units of theCRISPR-arrays identified by metagenomics were reduced to a singlerepeat-spacer-repeat unit, amenable to stuffer-spacer exchange byGG-assembly (AarI-restriction sites). Subsequently, CasΦ gene sequenceswere subcloned by GG-assembly into pRSFDuet-1 (Novagen) within MCSIwithout tags for efficiency of transformation plasmid interferenceassays, or fused to a C-terminal hexa-histidine tag for proteinpurification. For plasmid interference assays, mini-CRISPR arrays(repeat-spacer-repeat, or repeat-spacer-HDV ribozyme) amenable tostuffer-spacer exchange by GG-assembly (AarI-restriction sites) werecloned into MCS II of pRSFDuet. For genome editing experiments in humancells, casΦ genes were ordered as G-blocks from IDT encoding codonoptimized genes for expression in human cells. G-blocks were cloned viaGG-assembly into the vector backbone of pBLO62.5, downstream fused totwo SV40 NLSs via a GSG linker encoding sequence. The guide encodingsequence of pBLO62.5 was exchanged to encode for a single CRISPR-repeatof the respective homologue, followed by a 20 bp stuffer spacer sequenceamenable to GG-assembly exchange using the restriction enzyme SapI. Alist of plasmids and a brief description is given in FIG. 34 (providingTable 3). Plasmid sequences and maps will be made available on addgene.To reprogram the CasΦ vectors to target different loci, stuffer-spacerwere exchanged via GG-assembly to encode the guide for the selectedtarget site (guide spacer sequences are listed in FIG. 35 (providingTable 4)). Mutations in the casΦ genes were introduced by GG-assembly tocreate dcasΦ genes.

PAM Depletion DNA Interference Assay

PAM depletion assays were performed with both, CasΦ plasmids that eithercarried the whole CasΦ locus as derived from metagenomics (pPP049,pPP056 and pPP062), or with plasmids that contained only the casΦ geneand a mini CRISPR (pPP097, pPP102 and pPP107). Assays were performed asthree individual biological replicates. Plasmids containing casΦ andmini CRISPRs were transformed into E. coli BL21(DE3) (NEB) andconstructs containing CasΦ genomic loci were transformed into E. coliDH5α (QB3-Macrolab, UC Berkeley). Subsequently, electrocompetent cellswere prepared by ice cold H₂O and 10% glycerol washing. A plasmidlibrary was constructed with 8 randomized nucleotides upstream (5′) endof the target sequence. Competent cells were transformed in triplicateby electroporation with 200 ng library plasmids (0.1 mm electroporationcuvettes (Bio-Rad) on a Micropulser electroporator (Bio-Rad)). After atwo-hour recovery period, cells were plated on selective media andcolony forming units were determined to ensure appropriate coverage ofall possible combinations of the randomized 5′ PAM region. Strains weregrown at 25° C. for 48 hours on media containing appropriate antibiotics(either 100 μg/mL carbenicillin and 34 μg/mL chloramphenicol, or 100μg/mL carbenicillin and 50 μg/mL kanamycin) and 0.05 mMisopropyl-β-D-thiogalactopyranoside (IPTG), or 200 nManhydrotetracycline (aTc), depending on the vector to ensure propagationof plasmids and CasΦ effector production. Subsequently, propagatedplasmids were isolated using a QIAprep Spin Miniprep Kit (Qiagen).

PAM Depletion Sequencing Analysis

Amplicon sequencing of the targeted plasmid was used to identify PAMmotifs that are preferentially depleted. Sequencing reads were mapped tothe respective plasmids and PAM randomized regions were extracted. Theabundance of each possible 8 nucleotide combination was counted from thealigned reads and normalized to the total reads for each sample.Enriched PAMs were computed by calculating the log ratio compared to theabundance in the control plasmids, and were used to produce sequencelogos.

RNA Preparation for RNAseq

Plasmids containing CasΦ loci were transformed into chemically competentE. coli DH5α (QB3-Macrolab, UC Berkeley). Preparations were performed asthree individual biological replicates. Single colonies were picked toinoculate 5 mL starter cultures (LB, 34 μg/mL chloramphenicol) whichwere incubated at 37° C. shaking vigorously overnight. The next morning,main cultures were inoculated 1:100 (LB, 34 μg/mL chloramphenicol) andlocus expression was induced with 200 nM aTc for 24 h at 16° C. Cellswere harvested by centrifugation, resuspended in lysis buffer (20 mMHepes-Na pH 7.5 RT, 200 mM NaCl) and lysed using glass beads (0.1 mmglass beads, 4×30 s vortex at 4° C., interspaced by 30 s cool-down onice). 200 μL cell lysis supernatant were transferred into Trizol® forRNA extraction according to the manufacturer's protocol (Ambion). 10 μgRNA were treated with 20 units of T4-PNK (NEB) for 6 h at 37° C. for2′-3′-dephosphorylation. Subsequently, 1 mM ATP was added and the samplewas incubated for 1 h at 37° C. for 5′-phosphorylation before heatinactivation at 65° C. for 20 min and subsequent Trizol® purification.

RNA Analysis by RNAseq

cDNA libraries were prepared using the RealSeq-AC miRNA library kitillumina sequencing (somagenics). cDNA libraries were subjected toIllumina MiSeq® sequencing, and raw sequencing data was processed toremove adapters and sequencing artifacts, and high-quality reads weremaintained. The resulting reads were mapped to their respective plasmidsto determine the CRISPR locus expression and crRNA processing, andcoverage was calculated at each region.

Efficiency of Transformation Plasmid Interference Assay

CasΦ vectors were transformed into chemically competent E. coliBL21(DE3) (NEB). Individual colonies for biological replicates werepicked to inoculate three 5 mL (LB, Kanamycin 50 μg/mL) starter culturesto prepare electrocompetent cells the following day. 50 mL (LB,Kanamycin 50 μg/mL) main cultures were inoculated 1:100 and grownvigorously shaking at 37° C. to an OD₆₀₀ of 0.3. Subsequently, thecultures were cooled to room temperature and casΦ expression was inducedwith 0.2 mM IPTG. Cultures were grown to an OD₆₀₀ of 0.6-0.7 at 25° C.,before preparation of electrocompetent cells by repeated ice-cold H₂Oand 10% glycerol washes. Cells were resuspended in 250 μL 10% glycerol.90 μL aliquots were flash frozen in liquid nitrogen and stored at −80°C. The next day, 80 μL competent cells were combined with 3.2 μL plasmid(20 ng/μL pUC19 target plasmid, or 20 ng/μL pYTK001 control plasmid),incubated for 30 min on ice and split into three individual 25 μLtransformation reactions. After electroporation in 0.1 mmelectroporation cuvettes (Bio-Rad) on a Micropulser electroporator(Bio-Rad), cells were recovered in 1 mL recovery medium (Lucigen)supplemented with 0.2 mM IPTG, shaking at 37° C. for one hour.Subsequently, 10-fold dilution series were prepared and 5 μL of therespective dilution steps were spot-plated on LB-Agar containing theappropriate antibiotics. Plates were incubated overnight at 37° C. andcolonies were counted the following day to determine the transformationefficiency. To assess the transformation efficiency, the mean andstandard deviations were calculated from the cell forming units per ngtransformed plasmids for the electroporation triplicates.

Protein Production and Purification

CasΦ overexpression vectors were transformed into chemically competentE. coli BL21(DE3)-Star (QB3-Macrolab, UC Berkeley) and incubatedovernight at 37° C. on LB-Kan agar plates (50 μg/mL Kanamycin). Singlecolonies were picked to inoculate 80 mL (LB, Kanamycin 50 μg/mL) startercultures which were incubated at 37° C. shaking vigorously overnight.The next day, 1.5 L TB-Kan medium (50 μg/mL Kanamycin) were inoculatedwith 40 mL starter culture and grown at 37° C. to an OD₆₀₀ of 0.6,cooled down on ice for 15 min and gene expression was subsequentlyinduced with 0.5 mM IPTG followed by incubation overnight at 16° C.Cells were harvested by centrifugation and resuspended in wash buffer(50 mM HEPES-Na pH 7.5 RT, 1 M NaCl, 20 mM imidazole, 5% glycerol and0.5 mM TCEP), subsequently lysed by sonication, followed by lysateclarification by centrifugation. The soluble fraction was loaded on a 5mL Ni-NTA Superflow Cartridge (Qiagen) pre-equilibrated in wash buffer.Bound proteins were washed with 20 column volumes (CV) wash buffer andsubsequently eluted in 5 CV elution buffer (50 mM HEPES-Na pH 7.5 RT,500 mM NaCl, 500 mM imidazole, 5% glycerol and 0.5 mM TCEP). The elutedproteins were concentrated to 1 mL before injection into a HiLoad 16/600Superdex® 200 pg column (GE Healthcare) pre-equilibrated insize-exclusion chromatography buffer (20 mM HEPES-Na pH 7.5 RT, 500 mMNaCl, 5% glycerol and 0.5 mM TCEP). Peak fractions were concentrated to1 mL and concentrations were determined using a NanoDrop® 8000Spectrophotometer (Thermo Scientific). Proteins were purified at aconstant temperature of 4° C. and concentrated proteins were kept on iceto prevent aggregation, snap frozen in liquid nitrogen and stored at−80° C. AsCas12a was purified as previously described (Knott et al.(2019) Nat. Struct. Mol. Biol. 26:315).

In Vitro Cleavage Assays—Spacer Tiling

Plasmid targets were cloned by GG-assembly of spacer 2, found in theCRISPR-array of CasΦ-1, downstream to a cognate 5′-TTA PAM, ornon-cognate 5′-CCA PAM into pYTK095 (Target sequences are given in FIG.36 (providing Table 5)). Supercoiled plasmids were prepared bypropagation of the plasmid overnight at 37° C. in E. coli Mach1(QB3-Macrolab, UC Berkeley) in LB and Carbenicillin (100 μg/mL) andsubsequent preparation using a Qiagen Miniprep kit (Qiagen). Linear DNAtargets were prepared by PCR from the plasmid target. crRNA guides wereordered as synthetic RNA oligos from IDT (FIG. 37 (providing Table 6)),dissolved in DEPC H₂O and heated for 3 min at 95° C. before cool down atRT. Active RNP complexes were assembled at a concentration of 1.25 μM bymixing protein and crRNA (IDT) in a 1:1 molar ratio in cleavage buffer(10 mM Hepes-K pH 7.5 RT, 150 mM KCl, 5 mM MgCl₂, 0.5 mM TCEP) andincubation at RT for 30 min. Cleavage reactions were initiated byaddition of DNA (10 nM) to preformed RNP (1 μM) in reaction buffer (10mM Hepes-K pH 7.5 RT, 150 mM KCl, 5 mM MgCl₂, 0.5 mM TCEP). Thereactions were incubated at 37° C., quenched with 50 mM EDTA and storedin liquid nitrogen. Samples were thawed and treated with 0.8 unitsproteinase K (NEB) for 20 min at 37° C. Loading dye was added (GelLoading Dye Purple 6X, NEB) and samples were analyzed by electrophoresison a 1% agarose gel and stained with SYBR Safe (Thermo FisherScientific). For comparison to cleavage products, supercoiled plasmidswere digested with PciI (NEB) for linearization and Nt.BstNBI (NEB) forplasmid nicking and open circle formation. Comparable cleavage assaysunder varied conditions (n≥3) showed consistent results.

In Vitro Cleavage Assays—Radiolabeled Nucleic Acids

Active CasΦ RNP complexes were assembled in a 1:1.2 molar ratio bydiluting CasΦ protein to 4 μM and crRNA (IDT) to 5 μM in RNP assemblybuffer (20 mM HEPES-Na pH 7.5 RT, 300 mM KCl, 10 mM MgCl₂, 20% glycerol,1 mM TCEP) and incubation for 30 min at RT. Substrates were5′-end-labelled using T4-PNK (NEB) in the presence of ³²P-γ-ATP(Substrate sequences are given in FIG. 36 (providing Table 5)).Oligo-duplex targets were generated by combining ³²P-labelled andunlabelled complementary oligonucleotides in a 1:1.5 molar ratio. Oligoswere hybridized to a DNA-duplex concentration of 50 nM in hybridizationbuffer (10 mM Tris-Cl pH 7.5 RT, 150 mM KCl), by heating for 5 min to95° C. and a slow cool down to RT in a heating block. Cleavage reactionswere initiated by combining 200 nM RNP with 2 nM substrate in reactionbuffer (10 mM HEPES-Na pH 7.5 RT, 150 mM KCl, 5 mM MgCl₂, 10% glycerol,0.5 mM TCEP) and subsequently incubated at 37° C. For trans-cleavageassays, guide complementary activator substrates were diluted inoligonucleotide hybridization buffer (10 mM Tris pH 7.8 RT, 150 mM KCl)to a concentration of 4 μM, heated to 95° C. for 5 min, and subsequentlycooled down at RT to allow duplex formation for double strandedactivator substrates. Cleavage reactions were set up by combining 200 nMRNP with 100 nM activator substrate and incubation for 10 min at RTbefore addition of 2 nM ssDNA, or ssRNA, trans cleavage substrates.Reactions were stopped by addition of two volumes formamide loadingbuffer (96% formamide, 100 μg/mL bromophenol blue, 50 μg/mL xylenecyanol, 10 mM EDTA, 50 μg/mL heparin), heated to 95° C. for 5 min, andcooled down on ice before separation on a 12.5% denaturing urea-PAGE.Gels were dried for 4 h at 80° C. before phosphor-imaging visualizationusing an Amersham Typhoon™ scanner (GE Healthcare). Technical replicates(n≥2) and comparable cleavage assays under varied conditions (n≥3) ofbiological replicates (n≥2) showed consistent results. Bands werequantified using ImageQuant™ TL (GE) and cleaved substrate wascalculated from the intensity relative to the intensity observed at t=0min. Curves were fit to a One-Phase-Decay model in Prism 8 (graphpad) toderive the rate of cleavage.

In Vitro Pre-crRNA Processing Assay

Pre-crRNA substrates were 5′-end-labelled using T4-PNK (NEB) in thepresence of ³²P-7-ATP (Substrate sequences are given in FIG. 36(providing Table 5)). Processing reactions were initiated by combining50 nM CasΦ with 1 nM substrate in pre-crRNA processing buffer (10 mMTris pH 8 RT, 200 mM KCl, 5 mM MgCl₂ or 25 mM EDTA, 10% glycerol, 1 mMDTT) and subsequently incubated at 37° C. Substrate hydrolysis ladderswere prepared using the alkaline hydrolysis buffer according to themanufacturer's protocol (Ambion). 10 μL of the processing reactionproducts were treated with 10 units T4-PNK (NEB) for 1 h at 37° C. inthe absence of ATP for termini chemistry analysis. Reactions werestopped by addition of two volumes formamide loading buffer (96%formamide, 100 μg/mL bromophenol blue, 50 μg/mL xylene cyanol, 10 mMEDTA, 50 μg/mL heparin), heated to 95° C. for 3 min, and cooled down onice before separation on a 12.5%, or 20%, denaturing urea-PAGE. Gelswere dried for 4 h at 80° C. before phosphor-imaging visualization usingan Amersham Typhoon™ scanner (GE Healthcare). Technical replicates (n≥3)and comparable cleavage assays under varied conditions (n≥3) ofbiological replicates (n≥2) showed consistent results. Bands werequantified using ImageQuant™ TL (GE) and processed RNA was calculatedfrom the intensity at t=60 min relative to the intensity observed at t=0min.

Analytical Size Exclusion Chromatography

500 μL samples (5-10 μM protein, RNA, or reconstituted RNPs) wereinjected onto a S200 XK10/300 size exclusion chromatography (SEC) column(GE Healthcare) pre-equilibrated in SEC buffer (20 mM HEPES-Cl pH 7.5RT, 250 mM KCl, 5 mM MgCl₂, 5% glycerol and 0.5 mM TCEP). Prior to SEC,CasΦ RNP complexes were assembled by incubating CasΦ protein andpre-crRNA for 1 h in 2× pre-crRNA processing buffer (20 mM Tris pH 7.8RT, 400 mM KCl, 10 mM MgCl₂, 20% glycerol, 2 mM DTT).

Genome Editing in Human Cells

The GFP HEK293 reporter cells were generated via lentiviral integrationas previously described. Richardson et al, (2016) Nat. Biotechnol.34:339. Cells were routinely tested for absence of Mycoplasma using theMycoAlert Mycoplasma Detection Kit (Lonza), according to themanufacturer's protocol. GFP HEK293 reporter cells were seeded into96-well plates and transfected at 60-70% confluency the next dayaccording to the manufacturer's protocol with Lipofectamine® 3000 (LifeTechnologies) and 200 ng of plasmid DNA encoding the CasΦ gRNA andCasΦ-P2A-PAC fusion. As a comparison control, 200 ng of plasmid DNAencoding the SpyCas9 sgRNA and SpyCas9-P2A-PAC fusion was transfectedidentically, with target sequences adjusted for PAM differences. 24hours post-transfection, successfully transfected cells were selectedfor by adding 1.5 ng/mL puromycin to the cell culture media for 72hours. Cells were passaged regularly to maintain sub-confluentconditions and then analyzed on an Attune NxT Flow Cytometer with anautosampler. Cells were analyzed on the flow cytometer after 10 days toallow for clearance of G-FP from cells.

Results

Cas12J, or simply CasΦ as homage to its phage-restricted origin, is apreviously unknown family of Cas proteins encoded in the BiggiephageGlade. CasΦ contains a C-terminal RuvC domain with remote homology tothat of the TnpB nuclease superfamily from which type V CRISPR-Casproteins are thought to have evolved (FIG. 20). However, CasΦ shares <7%amino acid identity with other type V CRISPR-Cas proteins and is mostclosely related to a TnpB group distinct from miniature type V (Cas14)proteins (FIG. 19A).

CasΦ's unusually small size of ˜70-80 kDa, about half the size of theRNA-guided DNA cutting enzymes Cas9 and Cas12a (FIG. 19B), and its lackof co-occurring genes raised the question of whether CasΦ functions as abona fide CRISPR-Cas system. Three different CasΦ orthologs frommetagenomic assemblies were selected for study based on divergence oftheir protein and CRISPR repeat sequences (FIG. 21), referred to in FIG.21 as CasΦ-1, CasΦ-2 and CasΦ-3. To investigate the ability of CasΦ torecognize and target DNA in bacterial cells, it was tested whether thesesystems could protect Escherichia coli from plasmid transformation.CRISPR-Cas systems are known to target DNA sequences following orpreceding a 2-5 nucleotide Protospacer Adjacent Motif (PAM) forself-versus-non-self discrimination (Gleditzsch et al. (2019) RNABiology 16:504). To determine whether CasΦ uses a PAM, a library ofplasmids containing randomized regions adjacent to crRNA-complementarytarget sites was transformed into E. coli, thereby preferentiallydepleting plasmids including functional PAMs. This revealed thecrRNA-guided double-strand DNA (dsDNA) targeting capability of CasΦ anddistinct T-rich PAM sequences, including a minimal 5′-TBN-3′ PAMobserved for CasΦ-2 (FIG. 19C).

The E. coli expression system and plasmid interference assay was used todetermine the components required for CRISPR-CasΦ system function.RNA-sequencing analysis revealed transcription of the casΦ gene andCRISPR array but no evidence of other non-coding RNA such as atrans-activating CRISPR RNA (tracrRNA) encoded in or near the locus(FIG. 19D). In addition, it was found that CasΦ activity could bereadily directed against other plasmid sequences by altering the guideRNA, demonstrating the programmability of this system (FIG. 22A-22C).These findings suggest that in its native environment, CasΦ is afunctional phage protein and bona fide CRISPR-Cas effector capable ofcleaving DNA bearing complementarity to different crRNAs, likely otherMGEs, to abrogate superinfection (FIG. 19E). Furthermore, these resultsdemonstrate that this single-RNA system is much more compact than otheractive CRISPR-Cas systems (FIG. 19F).

CRISPR-Cas effector complexes identify and cleave foreign nucleic acidsduring the final stage of CRISPR-Cas mediated immunity against MGEs(Hille et al. (2018) Cell 172:1239). To determine how CasΦ achievesRNA-guided DNA targeting for Biggiephages, the recognition and cleavagerequirements of CasΦ in vitro were investigated. RNA-seq revealed thatthe spacer sequence within the crRNA, which is complementary to DNAtargets, is between 14-20 nucleotides (nt) long (FIG. 19D). Incubationof purified CasΦ (FIG. 24A-24D) with crRNAs of different spacer sizesalong with supercoiled plasmid or linear dsDNA revealed that target DNAcleavage requires the presence of a cognate PAM and a spacer of ≥14 nt(FIG. 23A; FIG. 25A). Analysis of the cleavage products showed that CasΦgenerates staggered 5′-overhangs of 8-12 nt (FIGS. 23B and 23C; FIGS.25B and 25C), similar to the staggered DNA cuts observed for other typeV CRISPR-Cas enzymes including Cas12a and CasX (Zetsche et al. (2015)Cell 163:759; Liu et al. (2019) Nature 566:218). It was observed thatCasΦ-2 and CasΦ-3 were more active in vitro than CasΦ-1, and thenon-target strand (NTS) was cleaved faster than the target-strand (TS)(FIG. 23D; FIG. 26A; FIGS. 27A and 27B). Furthermore, CasΦ was found tocleave ssDNA but not ssRNA targets (FIG. 26B), suggesting that CasΦ mayalso target ssDNA MGEs or ssDNA intermediates.

To assess the role of the RuvC domain in CasΦ-catalyzed DNA cleavage,the active site was mutated (D371A, D394A, or D413A) to produce a CasΦvariant (dCasΦ) that was found not to cleave dsDNA, ssDNA or ssRNA invitro (FIGS. 26A and 26B). When expressed in E. coli along with theCRISPR array, dCasΦ could not prevent transformation of acrRNA-complementary plasmid, consistent with a requirement forRuvC-catalyzed DNA cutting (FIG. 22A-22B). This observation, togetherwith the delayed cleavage of the target strand after non-target strandcleavage (FIG. 23D; FIGS. 27A and 27B), suggests that CasΦ cleaves eachstrand sequentially within the RuvC active site. Sequential dsDNA strandcleavage is consistent with the dsDNA cutting mechanism of the type VCRISPR-Cas proteins (10) that share closest evolutionary origin withCasΦ.

Furthermore, like other type V CRISPR-Cas effectors, CasΦ was found todegrade ssDNA in trans when activated by target dsDNA or ssDNA bindingin cis. Trans single-stranded DNAse, but not RNAse, activity upon DNAtarget recognition in cis was observed (FIG. 28A-28B). Thistrans-cleavage activity, coupled with a minimal PAM requirement, may beuseful for broader nucleic acid detection.

To provide genome defense, CRISPR-CasΦ systems must produce mature crRNAtranscripts to guide foreign DNA cleavage. Other type V CRISPR-Casproteins process their own pre-crRNAs using an internal active sitedistinct from the RuvC domain (Fonfara et al. Nature. 532, 517-521(2016)) or by recruiting Ribonuclease III to cleave a duplex RNAsubstrate formed by pre-crRNA base pairing with a tracrRNA (Burstein etal. (2017) Nature 542:237; Harrington et al. (2018) Science 362:839; Yanet al. (2019) Science 363:88; Shmakov et al. (2015) Mol. Cell. 60:385).The absence of a detectable tracrRNA encoded in CRISPR-CasΦ genomic locihinted that CasΦ may catalyze crRNA maturation on its own. To test thispossibility, purified CasΦ was incubated with substrates designed tomimic the pre-crRNA structure (FIG. 29A). Reaction productscorresponding to a 26-29 nucleotide-long repeat and 20 nucleotide guidesequence of the crRNA were observed only in the presence of wildtypeCasΦ, corroborated by RNA-seq analysis of native loci (FIG. 19D; FIG.29A; FIG. 29C; FIG. 30A-30C). In control experiments, it was found thatCasΦ-catalyzed pre-crRNA processing is magnesium-dependent (FIG. 29B;FIG. 30A-30C), which is different from all other known CRISPR-Cas RNAprocessing reactions and suggested a distinct chemical mechanism ofcleavage. Notably, the RuvC domain itself employs a magnesium-dependentmechanism to cleave DNA substrates (Nowotny et al. (2009) EMBO Rep.10:144), and some RuvC domains have been reported to haveendoribonucleolytic activity (Yan et al. (2019) Science 363:88). Basedon these observations, a CasΦ containing a RuvC-inactivating mutationwas tested; it was found to be incapable of processing pre-crRNAs (FIG.29B; FIGS. 30A and 30B). Both wild-type and catalytically inactivatedCasΦ proteins are capable of crRNA binding, and their reconstitutedcomplexes with pre-crRNA have similar elution profiles from a sizeexclusion column, suggesting no pre-crRNA binding or protein stabilitydefect resulting from the RuvC point mutation (FIG. 31A-31B).

It was hypothesized that if the CasΦ RuvC domain is responsible forpre-crRNA cleavage, the products should contain 5′-phosphate and 2′- and3′-hydroxyl moieties as observed in RNAs generated by the RuvC-relatedRNase HI enzymes (Nowotny et al. (2009) supra). In contrast, other typeV CRISPR-Cas enzymes process pre-crRNA by a metal-independent acid-basecatalysis mechanism in an active site distinct from the RuvC domain,generating 2′-3′-cyclic phosphate crRNA termini, as observed for Cas12a(Swarts et al. (2017) Mol. Cell. 66:221). PNK phosphatase treatment ofCasΦ-generated crRNA followed by denaturing acrylamide gel analysisshowed no change in the crRNA migration behavior, distinct from thechange in mobility detected in a similar experiment conducted with crRNAgenerated by Cas12a (FIG. 29C; FIG. 30C). This result implies that no2′-3′-cyclic phosphate was formed during the reaction catalyzed by CasΦ,in contrast to the RuvC-independent acid-base catalyzed pre-crRNAprocessing reaction by AsCas12a (FIGS. 29C and 29D). Together, thesedata demonstrate that CasΦ uses a single active site for both pre-crRNAprocessing and DNA cleavage, which is a previously unseen activity for aRuvC active site or a CRISPR-Cas enzyme.

The versatility and programmability of CRISPR-Cas systems have sparked arevolution in biotechnology and fundamental research, as they have beenemployed to manipulate genomes of virtually any organism. To investigatewhether the DNA cleavage activity of CasΦ can be harnessed forprogrammed human genome editing, a gene disruption assay was performed(Liu et al. (2019) Nature 566:218; Oakes et al. (2016) Nat. Biotechnol.34:646) using CasΦ co-expressed with a suitable crRNA in HEK293 cells(FIG. 32A). It was found that CasΦ-2 and CasΦ-3, but not CasΦ-1, caninduce targeted disruption of a genomically integrated gene encodingenhanced green fluorescent protein (EGFP) (FIG. 33A; FIG. 32B). In onecase, CasΦ-2 with an individual guide RNA was able to edit up to 33% ofcells (FIG. 33A), comparable to levels initially reported forCRISPR-Cas9, CRISPR-Cas12a, and CRISPR-CasX (Zetsche et al. (2015) Cell163:759; Liu et al. (2019) supra; Mali et al. (2013) Science 339:823).The small size of CasΦ in combination with its minimal PAM requirementis particularly advantageous for both vector-based delivery into cellsand a wider range of targetable genomic sequences, providing a powerfuladdition to the CRISPR-Cas toolbox.

CasΦ represents a new family of CRISPR-Cas enzymes defined by its singleactive site for both RNA and DNA cutting. Three other well-characterizedCas enzymes Cas9, Cas12a, and CasX, use one (Cas12a and CasX) or twoactive sites (Cas9) for DNA cutting and rely on a separate active site(Cas12a) or additional factors (CasX and Cas9) for crRNA processing(FIG. 33B). The finding that in CasΦ a single RuvC active site iscapable of both crRNA processing and DNA cutting suggests that sizelimitations of phage genomes, possibly in combination with largepopulation sizes and higher mutation rates in phages compared toprokaryotes (24-26), led to a consolidation of chemistries within onecatalytic center.

FIG. 19A-19F. CasΦ is a bona fide CRISPR-Cas system from huge phages.(A) Maximum Likelihood phylogenetic tree of reported type V effectorproteins and respective predicted ancestral TnpB nucleases. Bootstrapand approximate likelihood-ratio test values ≥90 are denoted on thebranches with black circles. (B) Illustrations of the genomic loci ofCRISPR-Cas systems previously employed in genome editing applications.(C) Graphical representation of the PAM depletion assay and theresulting PAMs for three CasΦ orthologs. (D) RNA-sequencing results(left) mapped onto the native genomic loci of CasΦ orthologs and theirupstream and downstream non-coding regions as cloned into theirrespective expression plasmids. Enlarged view of RNA mapped onto thefirst repeat-spacer pair (right). (E) Schematic of the hypothesizedfunction of Biggiephage-encoded CasΦ in an instance of superinfection ofits host. CasΦ may be used by the huge phage to eliminate competingmobile genetic elements. (F) Predicted molecular weights of theribonucleoprotein (RNP) complexes of small CRISPR-Cas effectors andthose functional in editing of mammalian cells.

FIG. 20. Maximum likelihood phylogenetic tree of type V subtypes a-k.Bootstrap and approximate likelihood ratio test values >90 are shown onthe branches (circles).

FIG. 21. CasΦ crRNA repeats are highly diverse. A similarity matrix wasbuilt and visualized using a heatmap and hierarchical clusteringdendrogram. CasΦ-1, CasΦ-2, and CasΦ-3 repeats.

FIG. 22A-22C. CasΦ-3 protects against plasmid transformation. (A) Schemeillustrating the efficiency of transformation (EOT) assay. (B) EOT assayshowing that CasΦ, programmed by a beta-lactamase (bla) gene targetingguide, reduces the efficiency of pUC19 transformation. Experiments wereperformed in three biological replicates and technical electroporationtransformation triplicates (dots; n=3 each, mean±s.d.). Competent cellswere tested for general transformation efficiency (grey bars) bytransformation of pYTK095, which is not targeted by the tested bla andNT (non-targeting) guide. (C) EOT in dependence of CasΦ-3 RuvC activesite residue variation (RuvCI: D413A; RuvCII: E618A; RuvCIII: D708A).N=3 each, mean±s.d. Competent cells were tested for generaltransformation efficiency (grey bars).

FIG. 23A-23D. CasΦ cleaves DNA. (A) Supercoiled plasmid cleavage assayin dependence of the guide spacer length. (B) Cleavage assay targetingdsDNA oligo-duplices for mapping of the cleavage structure. (C) Schemeillustrating the cleavage pattern. (D) NTS and TS DNA cleavageefficiency (n=3 each, mean±s.d.). Data is shown in FIG. 27B.

FIG. 24A-24D. Purification of apo CasΦ. (A) SDS-PAGE of the purified apoCasΦ orthologs and their dCasΦ variants. (B) Analytical size-exclusionchromatography (S200) of CasΦ-1 WT and dCasΦ-1. (C) Analyticalsize-exclusion chromatography (S200) of CasΦ-2 WT and dCasΦ-2. D)Analytical size-exclusion chromatography (S200) of CasΦ-3 WT anddCasΦ-3.

FIG. 25A-25C. CasΦ targets DNA in vitro to produce staggered cuts. (A)Linear PCR-fragment cleavage assay in dependence of the guide spacerlength and presence of a cognate 5′-TTA-3′ PAM (left), or non-cognate5′-CCA-3′ PAM (right). (B) Cleavage assay targeting dsDNA oligo-duplicesfor mapping of the cleavage structure. (C) Scheme illustrating thecleavage pattern of the staggered cuts. Shown are the proposed R-loop(replication loop) structures formed by CasΦ upon target DNA binding tothe crRNA spacer.

FIG. 26A-26C. CasΦ targets dsDNA and ssDNA, but not RNA in vitro. (A)Cleavage assay assessing the ability of CasΦ and dCasΦ variant (D371A,D394A and D413A) RNPs to cleave the target strand (TS), and non-targetstrand (NTS), of a dsDNA oligo duplex. (B) Cleavage assay testing theability of CasΦ and dCasΦ variant (D371A, D394A and D413A) RNPs totarget and cleave a single stranded DNA, or RNA, target strand.

FIG. 27A-27B. Cleavage assay comparing TS and NTS cleavage efficiency byCasΦ. (A) Cleavage assay curves, fit to the One Phase Decay model usingPrism 8 (GraphPad) (n=3 each, mean±s.d.). Cleaved fractions arecalculated based on the substrate band intensities at t=(0 min) (panelB) relative to the respective time point. (B) Urea-Page gels of thethree independent reaction replicates (Replicates 1, 2 and 3). Thispanel also relates to FIG. 23D for CasΦ-2.

FIG. 28A-28B. CasΦ targets ssDNA, but not RNA, in trans upon activationin cis. (A) Cleavage assay comparing the trans cleavage activities ofCasΦ-1, CasΦ-2 and CasΦ-3 on ssDNA and ssRNA as targets in trans independence of either ssDNA, dsDNA, or ssRNA as activators in cis. (B)Cleavage assay comparing the trans cleavage activity of CasΦ-1, CasΦ-2and CasΦ-3.

FIG. 29A-29D. CasΦ processes pre-crRNA within the RuvC active site. (A)pre-crRNA substrates and processing sites (triangles)—as derived fromthe OH-ladder in panel C. (B) Pre-crRNA processing assay for CasΦ-1 andCasΦ-2 in dependence of Mg²⁺ and RuvC active site residue variation(D371A and D394A) (n=3 each, mean±s.d.; t=60 min). Data is shown in FIG.30B. (C) Left and middle: Alkaline hydrolysis ladder (OH) of thepre-crRNA substrate. Right: PNK-phosphatase treatment of the CasΦ andCas12a cleavage products. (D) Graphical representation of the maturecrRNA termini chemistry of CasΦ and Cas12a and PNK-phosphorylasetreatment outcomes.

FIG. 30A-30C. CasΦ-1 and CasΦ-2, but not CasΦ-3, process pre-crRNA. (A)Pre-crRNA processing assay for CasΦ-1, CasΦ-2 and CasΦ-3 in dependenceof Mg²⁺ and RuvC active site catalytic residues (dCasΦ variants). (A)Processing reaction replicates for CasΦ-1 and CasΦ-2 at t=0 min and t=60min. Squares indicate quantified bands. This panel relates to FIG. 29B.(C) Pre-crRNA processing assay for CasΦ-1, CasΦ-2 and AsCas12a independence of Mg²⁺ and RuvC active site catalytic residues (dCasΦvariants).

FIG. 31A-31B. CasΦ WT and dCasΦ proteins form RNPs with pre-crRNA. (A)Analytical size-exclusion chromatography (S200) of wild-type proteins,pre-crRNA, and their respective reconstituted RNP. (B) Analyticalsize-exclusion chromatography (S200) of dCasΦ variant proteins,pre-crRNA, and their respective reconstituted RNP.

FIG. 32A-32C. CasΦ mediated EGFP gene disruption in HEK293 cells. (A)Schematic of the experimental workflow of the GFP disruption assay(left) and EGFP disruption by SpyCas9 (right) (B) CasΦ guides with GFPdisruption below 5% (n=3 each, mean±s.d.). (C) EGFP map showing thetarget sites and orientation of guides (arrows and numbers). Yellowtriangles indicate the best guides for gene disruption (relates to FIG.34A). Guide sequences are listed in Table 4 (presented in FIG. 35).

FIG. 33A-33B. CasΦ is functional for human genome editing. (A) GFPdisruption using CasΦ-2 (left) and CasΦ-3 (right) and a non-targeting(NT) guide as a negative control (n=3 each, mean±s.d.). All testedguides and targeted regions within the EGFP gene are shown in FIG.32A-32C. (B) Scheme illustrating the differences in RNA processing andDNA cutting for Cas9, Cas12a, CasX, and CasΦ.

FIG. 34 presents Table 3.

FIG. 35 presents Table 4.

FIG. 36 presents Table 5.

FIG. 37 presents Table 6.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A eukaryotic cell comprising: a) a nucleasecomprising an amino acid sequence that is at least 98% identical to theamino acid sequence set forth in SEQ ID NO:120; b) a target nucleic acidcomprising a target strand and a non-target strand; and c) a recombinantguide nucleic acid comprising in a 5′ to 3′ orientation: i) a firstregion that binds to the nuclease; and ii) a second region that iscomplementary to a portion of the target strand of the target nucleicacid, wherein the target nucleic acid comprises a protospacer adjacentmotif (PAM) of 5′-NTTN-3′, wherein T is thymine and N is any nucleotide,and wherein the PAM is on the non-target strand of the target nucleicacid.
 2. The eukaryotic cell of claim 1, wherein the nuclease comprisesan amino acid sequence that is at least 99% identical to the amino acidsequence of SEQ ID NO:
 120. 3. The eukaryotic cell of claim 1, whereinthe nuclease comprises an amino acid sequence that is 100% identical tothe amino acid sequence of SEQ ID NO:
 120. 4. The eukaryotic cell ofclaim 1, wherein the nuclease comprises at least one amino acid sequenceselected from: a) EEACKKFVREN b) GPAIANIIAKSREFTEW c) LNLIIKNAVN d)VKVDNKNKNNL e) RKNEIAKLNGEQE f) GYLLQKPSPNKSIYCY g) PGYVPKWQY h)CVFDMRGLLRT i) TVDVGQNNP j) GEKFNCLKCG,  and k) VAITAQSMPKP.


5. The eukaryotic cell of claim 1, wherein the first region of therecombinant guide nucleic acid comprises a nucleotide sequence that isat least 80% identical to SEQ ID NO:181.
 6. The eukaryotic cell of claim1, wherein the first region of the recombinant guide nucleic acidcomprises a nucleotide sequence that is at least 95% identical to SEQ IDNO:181.
 7. The eukaryotic cell of claim 1, wherein a nuclearlocalization signal (NLS) is fused via an amide bond to the N terminusof the nuclease, the C-terminus of the nuclease, or both termini of thenuclease.
 8. The eukaryotic cell of claim 7, wherein the NLS comprisesthe amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO:50. 9.The eukaryotic cell of claim 1, wherein said eukaryotic cell comprises aDNA donor template.
 10. The eukaryotic cell of claim 1, wherein the PAMcomprises the sequence 5′-VTTN-3′, wherein T is thymine, N is anynucleotide, and V is selected from adenine, guanine, and cytosine. 11.The eukaryotic cell of claim 1, wherein the nuclease is fused to aheterologous polypeptide.
 12. The eukaryotic cell of claim 1, whereinthe eukaryotic cell is a human cell.
 13. The eukaryotic cell of claim 1,wherein the eukaryotic cell is a mammalian cell.
 14. The eukaryotic cellof claim 1, wherein the eukaryotic cell is a hematopoietic stem cell.15. The eukaryotic cell of claim 1, wherein the eukaryotic cell is a Tcell.
 16. A population of cells comprising the eukaryotic cell ofclaim
 1. 17. A eukaryotic cell comprising A) a target nucleic acidcomprising a target strand and a non-target strand, wherein the targetnucleic acid comprises a protospacer adjacent motif (PAM) of 5′-NTTN-3′,wherein T is thymine and N is any nucleotide, and wherein the PAM is onthe non-target strand of the target nucleic acid; and B) at least onenucleic acid molecule encoding: a) a nuclease comprising an amino acidsequence that is at least 98% identical to the amino acid sequence setforth in SEQ ID NO:120; and b) a recombinant guide nucleic acidcomprising in a 5′ to 3′ orientation: i) a first region that binds tothe nuclease; and ii) a second region that is complementary to a portionof the target strand of the target nucleic acid.
 18. The eukaryotic cellof claim 17, wherein the nuclease comprises an amino acid sequence thatis at least 99% identical to the amino acid sequence of SEQ ID NO: 120.19. The eukaryotic cell of claim 17, wherein the nuclease comprises anamino acid sequence that is 100% identical to the amino acid sequence ofSEQ ID NO:
 120. 20. The eukaryotic cell of claim 17, wherein thenuclease comprises at least one amino acid sequence selected from: (a)EEACKKFVREN (b) GPAIANIIAKSREFTEW (c) LNLIIKNAVN (d) VKVDNKNKNNL (e)RKNEIAKLNGEQE (f) GYLLQKPSPNKSIYCY (g) PGYVPKWQY (h) CVFDMRGLLRT (i)TVDVGQNNP (j) GEKFNCLKCG,  and (k) VAITAQSMPKP.


21. The eukaryotic cell of claim 17, wherein the nucleic acid moleculeis an expression vector.
 22. The eukaryotic cell of claim 21, whereinthe expression vector is an adeno-associated viral vector.
 23. Theeukaryotic cell of claim 17, wherein the eukaryotic cell comprises a DNAdonor template.
 24. The eukaryotic cell of claim 17, wherein the nucleicacid molecule is associated with a lipid or liposome.
 25. The eukaryoticcell of claim 17, wherein the nuclease is fused to a heterologouspolypeptide.
 26. The eukaryotic cell of claim 17, wherein the eukaryoticcell is selected from the group consisting of a mammalian cell, a humancell, a T cell, and a hematopoietic stem cell.
 27. A population of cellscomprising the eukaryotic cell of claim
 17. 28. The eukaryotic cell ofclaim 1, wherein the nuclease comprises a RuvC nuclease domain.
 29. Theeukaryotic cell of claim 1, wherein the recombinant guide nucleic acidcomprises at least one modified sugar moiety.
 30. The eukaryotic cell ofclaim 29, wherein the at least one modified sugar moiety is 2′-O-Methyl.31. The eukaryotic cell of claim 1, wherein the recombinant guidenucleic acid comprises a phosphorothioate linkage.
 32. The eukaryoticcell of claim 17, wherein the nuclease comprises a RuvC nuclease domain.